Multifactorial Optimization of Contrast-Enhanced Nanofocus Computed Tomography for Quantitative Analysis of Neo-Tissue Formation in Tissue Engineering Constructs

To progress the fields of tissue engineering (TE) and regenerative medicine, development of quantitative methods for non-invasive three dimensional characterization of engineered constructs (i.e. cells/tissue combined with scaffolds) becomes essential. In this study, we have defined the most optimal staining conditions for contrast-enhanced nanofocus computed tomography for three dimensional visualization and quantitative analysis of in vitro engineered neo-tissue (i.e. extracellular matrix containing cells) in perfusion bioreactor-developed Ti6Al4V constructs. A fractional factorial ‘design of experiments’ approach was used to elucidate the influence of the staining time and concentration of two contrast agents (Hexabrix and phosphotungstic acid) and the neo-tissue volume on the image contrast and dataset quality. Additionally, the neo-tissue shrinkage that was induced by phosphotungstic acid staining was quantified to determine the operating window within which this contrast agent can be accurately applied. For Hexabrix the staining concentration was the main parameter influencing image contrast and dataset quality. Using phosphotungstic acid the staining concentration had a significant influence on the image contrast while both staining concentration and neo-tissue volume had an influence on the dataset quality. The use of high concentrations of phosphotungstic acid did however introduce significant shrinkage of the neo-tissue indicating that, despite sub-optimal image contrast, low concentrations of this staining agent should be used to enable quantitative analysis. To conclude, design of experiments allowed us to define the most optimal staining conditions for contrast-enhanced nanofocus computed tomography to be used as a routine screening tool of neo-tissue formation in Ti6Al4V constructs, transforming it into a robust three dimensional quality control methodology.     

Phosphotungstic Acid-Eosin Combined with Hematoxylin as A Stain for Negri Bodies in Paraffin Sections

Experimentation with the Papanicolaou stain in this laboratory led to the discovery that the eosin, combined with phosphotungstic acid, was responsible for differential staining of Negri bodies. Eosin prepared as in EA 36 was used, but without the light green and Bismarck brown. Paraffin sections of hippocampus from brains of animal affected with rabies were fixed in 10% formol or in a mixture of 2 volumes of saturated aqueous HgCl₂ and 1 volume of absolute alcohol. They were stained first with hematoxylin and then with eosin. This procedure gave better results than staining with other types of eosin or by the original EA 36 mixture. The Negri bodies were well stained and their structure easily visible. The best results were obtained from material fixed with the HgCl₂ solution.     

The role of phosphotungstic and phosphomolybdic acids in connective tissue staining I. Histochemical studies

Synopsis An investigation of the role of phosphotungstic and phosphomolybdic acids in Mallory-like trichrome methods showed unexpectedly that, rather than acting as mordants to anionic dyes, these polyacids selectively blocked staining of all tissue components other than connective tissue fibres to Aniline Blue and other similar fibrereactive dyes. Connective tissue components were found to contain residues resembling histidine that are easily accessible to anionic dyes. Blocking towards typical anionic dyes for demonstrating plasma proteins, such as Biebrich Scarlet, was also demonstrated but was less complete. The blockade of both types of dye was labile if the staining times were extended; plasma dyes were more sensitive than fibre dyes in this respect. Histochemical reactions for tyrosine residues were blocked. In connective tissue, phosphotungstic acid did not block histidine residues demonstrable by the coupled tetrazonium reaction with previous iodination. Thus it is postulated that differential trichrome staining occurs by binding of Aniline Blue to basic residues in the connective tissue not blocked by phosphotungstic acid and subsequent replacement of the blocking agent by an anionic dye. The binding of phosphotungstic acid to both epithelium and connective tissue was demonstrated by the quenching of autofluorescence in these regions and by the reduction of the bound PTA to blue coloured products with titanium trichloride.     

Application of the Papanicolaou Stain to Paraffin Sections

Routine paraffin sections from tissues fixed either in aqueous formalin, 80% alcohol (with or without 1% trichloracetic acid added), Carnoy's alcohol-chloroform-acetic (6:3:1) and Bouin's fixative were stained as follows: Harris' hematoxylin, 6 min; running water, 2-3 min; ascending grades of alcohol to 95%; orange G, 0.5% and phosphotungstic acid, 0.015% in 95% alcohol, 5 min; 95% alcohol, 2 changes; Papanicolaou's EA36, 2.5 min; dehydration, clearing, and covering in Permount. The results show morphology better than hematoxylin and eosin and the technic is recommended particularly for keratin, which always stains bright orange.     

The Effects of Ellagic Acid upon Brain Cells: A Mechanistic View and Future Directions

Ellagic acid (EA, 2,3,7,8-tetrahydroxy-chromeno; C₁₄H₆O₈) is a polyphenol derived from fruits (pomegranates, berries) and nuts. EA exhibits antioxidant capacity and induces anti-inflammatory actions in several mammalian tissues. EA has been characterized as a possible neuroprotective agent, but the number of reports is still limited to conclude whether and how EA exerts neuroprotection in humans. In this regard, performing additional studies considering the potential beneficial and/or toxicological roles for EA on brain cells would be an important step towards fully understanding of when and how EA may be securely utilized by humans as a neuroprotective agent. The aim of the present work is to discuss data related to the neuronal and glial effects of EA and the mechanisms underlying such events. Moreover, future directions are suggested as a potential guide to be utilized by researchers interested in investigating the neuronal and glial actions of EA hereafter.     

The protective role of ellagitannins flavonoids pretreatment against N-nitrosodiethylamine induced-hepatocellular carcinoma

Ellagitannins are esters of glucose with hexahydroxydiphenic acid; when hydrolyzed, they yield ellagic acid (EA), the dilactone of hexahydroxydiphenic acid. EA has been receiving the most attention, because it has potent antioxidant activity, radical scavenging capacity, chemopreventive and antiapoptotic properties. Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of liver, and accounts for as many as one million deaths worldwide in a year. The aim of the present study was to evaluate the antioxidant and chemopreventive efficiency of ellagic acid against N-nitrosodiethylamine (NDEA) induced hepatocarcinogenesis in rats. Rats were classified into four groups as follows: normal control group, group injected i.p. with a single dose (200 mg/kg b.wt.) of NDEA, third group daily administered orally EA with a dose of 50 mg/kg b.wt. for 7 days before and 14 days after NDEA administration, and fourth group received a similar dose of EA for 21 days after the dose of NDEA administration. The model of NDEA-injected hepatocellular carcinomic (HCC) rats elicited significant declines in liver antioxidant enzyme activities; glutathione peroxidase (GPX), gamma glutamyl transferase (γ-GT) and glutathione-S-transferase (GST), with a reduction in reduced glutathione (GSH) and serum total protein with concomitant significant elevations in tumor markers arginase and α-l-fucosidase, and liver enzymes; aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and glutathione-S-transferase (GST), glucose-6-phosphate dehydrogenase (G6PD), direct and total bilirubin. The oral administration of EA as a protective agent, produced significant increases in tested antioxidant enzyme activities and serum total protein concomitant with significant decreases in the levels of tumor markers arginase and α-l-fucosidase as well as liver enzymes, direct and total bilirubin. Similarly, the oral administration of EA, as a curative agent produced similar changes to those when EA was used as a protective agent, but to a lesser extent. In addition, it was noted that HCC rats exhibited a degree of DNA fragmentation; however, EA administration partially inhibited the DNA fragmentation. Therefore, EA has the ability to scavenge free radicals, prevent DNA fragmentation, reduce liver injury and protect against oxidative stress.     

Degradation and regurgitation of extracellular proteins by cultured mouse peritoneal macrophages and baby hamster kidney fibroblasts. Kinetic evidence that the transfer of proteins to lysosomes is not irreversible

The uptake and degradation of bovine serum albumin (BSA), bovine liver catalase, and rabbit muscle enolase have been studied in cultured mouse peritoneal macrophages (MPM) and baby hamster kidney fibroblasts (BHK cells). Rates constant for the uptake of the three proteins by MPM were similar. In addition, BSA accumulation was independent of BSA concentration in the uptake medium and was not inhibited by a large excess of serum, suggesting that protein accumulation was by fluid phase pinocytosis. Following an overnight uptake, 20-30% of the accumulated protein was subsequently regurgitated into the medium in a trichloroacetic acid/phosphotungstic acid-precipitable form. This material co-migrated with the authentic protein during molecular sieve chromatography on Sephadex G-50. The rates of appearance of trichloroacetic acid/phosphotungstic acid-insoluble products were greater than expected for cell death and leakage. The observed first order rate constants, kobs, for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and trichloroacetic acid/phosphotungstic acid-insoluble products in the culture medium were identical, indicating that both products were released in parallel from MPM and BHK cells. The kobs for intracellular BSA degradation and regurgitation were independent of the initial BSA concentration in the uptake medium, but were decreased about 35% when degradation was allowed to proceed in the presence of high concentrations of serum. Degradation was also inhibited by chloroquine and pepstatin. Inhibition of degradation was accompanied by an increase in the total amount of regurgitated protein appearing in the medium. Remarkably, however, these inhibitors also decreased kobs for regurgitation, thereby preserving the similarity in the observed rate constants for the appearance of trichloroacetic acid/phosphotungstic acid-soluble and trichloroacetic acid/phosphotungstic acid-insoluble products. These and other results were inconsistent with desorption of proteins from the surface of the culture dish or the surface of cells as the source of the trichloroacetic acid/phosphotungstic acid-insoluble label appearing in the medium.(ABSTRACT TRUNCATED AT 400 WORDS)     

Molecular characterization of the Bacillus anthracis main S-layer component: evidence that it is the major cell-associated antigen

Bacillus anthracis, the aetiological agent of anthrax, is a Gram-positive spore-forming bacterium. The cell wall of vegetative cells of B. anthracis is surrounded by an S-layer. An array remained when sap, a gene described as encoding an S-layer component, was deleted. The remaining S-layer component, termed EA1, is chromosomally encoded. The gene encoding EA1 (eag) was obtained on two overlapping fragments in Escherichia coli and shown to be contiguous to the sap gene. The EA1 amino acid sequence, deduced from the eag nucleotide sequence, shows classical S-layer protein features (no cysteine, only 0.1% methionine, 10% lysine, and a weakly acidic pi). Similar to Sap and other Gram-positive surface proteins, EA1 has three 'S-layer-homology’motifs immediately downstream from a signal peptide. Single- and double-disrupted mutants were constructed. EA1 and Sap were co-localized at the cell surface of the wild-type bacilli. However, EA1 was more tightly bound than Sap to the bacteria. Electron microscopy studies and in vivo experiments with the constructed mutants showed that EA1 constitutes the main lattice of the B. anthracis S-layer, and is the major cell-associated antigen.     

Study on the heterogeneous degradation of chitosan with hydrogen peroxide under the catalysis of phosphotungstic acid

The oxidative degradation of chitosan with H₂O₂ aqueous solution was carried out under the catalysis of phosphotungstic acid in heterogeneous phase. The optimal conditions of degradation were determined by orthogonal tests. The structure of the degraded product was characterized by Fourier-transform infrared spectra (FTIR), diffuse reflectance spectra (DRS) and X-ray diffraction (XRD) analysis. The mechanism of the degradation was correlated with cleavage of the glycosidic bond. The experimental results showed that chitosan can be effectively degraded with H₂O₂ under the catalysis of phosphotungstic acid.     

The axonal reticulum in the neurons of the superior cervical ganglion of the rat as a direct extension of the Golgi apparatus

Summary Adrenergic neurons from the superior cervical ganglion of the rat have been studied with the chromaffin reaction and the zinc iodide-osmium tetroxide method. Phosphotungstic acid staining at low pH and a combined acid phosphatase reaction and phosphotungstic acid staining have also been performed on glycolmethacrylate-embedded tissue. The results indicate that phosphotungstic acid-positive elements lacking acid phosphatase activity are present at the inner side of the Golgi apparatus. These elements give rise directly to reticular differentiations, carrying catecholamines, or to tubular extensions, representing the origin of the axonal reticulum. On these tubules, reticular differentiations can again be formed at any level. In the cell body, the differentiations are mainly found close to the neurolemma. In the axons, they are especially abundant at the axon terminals. Large granules may be associated with the reticular differentiations and small and large granules may detach from them.It is concluded that the whole catecholamine-producing and/or-storing system in sympathetic neurons can be considered as a direct extension of the Golgi apparatus, set up for local catecholamine synthesis. The relative importance of small and large granules along this system may reflect the functional status of the nerve cell.     

THE FINE STRUCTURE OF ELASTIC FIBERS

The fine structure of developing elastic fibers in bovine ligamentum nuchae and rat flexor digital tendon was examined. Elastic fibers were found to contain two distinct morphologic components in sections stained with uranyl acetate and lead. These components are 100 A fibrils and a central, almost amorphous nonstaining area. During development, the first identifiable elastic fibers are composed of aggregates of fine fibrils approximately 100 A in diameter. With advancing age, somewhat amorphous regions appear surrounded by these fibrils. These regions increase in prominence until in mature elastic fibers they are the predominant structure surrounded by a mantle of 100 A fibrils. Specific staining characteristics for each of the two components of the elastic fiber as well as for the collagen fibrils in these tissues can be demonstrated after staining with lead, uranyl acetate, or phosphotungstic acid. The 100 A fibrils stain with both uranyl acetate and lead, whereas the central regions of the elastic fibers stain only with phosphotungstic acid. Collagen fibrils stain with uranyl acetate or phosphotungstic acid, but not with lead. These staining reactions imply either a chemical or an organizational difference in these structures. The significance and possible nature of the two morphologic components of the elastic fiber remain to be elucidated.     

Protective effect of ellagic acid (EA) on micronucleus formation induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in mammalian cells, in in vitro assays and in vivo

The beneficial effects of fruits and vegetables with respect to age-related diseases such as diabetes, atherosclerosis and several types of cancer are widely recognized and confirmed by several epidemiological studies. A possible approach for evaluating the protective potential of promising diet constituents is to evaluate their beneficial effect with respect to a set of biomarkers that are indicative of a potential risk for developing degenerative diseases. Among the numerous biomarkers of the effect of food-related carcinogens and for the assessment of the degree of risk for disease, chromosomal damage detection is very predictive. The aim of this study was to test antigenotoxic effect of ellagic acid (EA) both in in vitro and in vivo studies, in combination with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), a methylating agent. EA, a naturally occurring and widely distributed plant phenol, has been intensively studied but with conflicting results, depending on the endpoints considered and the experimental material employed. In vitro and in vivo studies differ in their experimental schedule: in the in vitro study pre- and post-treatments and simultaneous treatments with EA were performed, while in the in vivo study only pre-treatment was carried out. The results of this study clearly demonstrate a protective action of EA with respect to MNNG-induced micronuclei and cell proliferation both in vitro and in vivo. The lack of effect in the post-treatment in in vitro experiments excludes a possible effect of EA on DNA-repair systems. On the other hand, consumption of EA can have a protective action against primary DNA damage induced by oxidative stress.     

System-forming functions of bound water in the mechanism of topochemical reactions of formation of ultrathin layers on water surface

The system-forming role of the surface layer of water in the process of formation of ultrathin layers from the epoxide oligomer and triethylenetetramine or phosphotungstic acid on the surface of the aqueous phase is shown. Previously, this process was studied experimentally. The surface layer of the aqueous phase plays the role of a matrix, on which an epoxide oligomer monolayer and triethylenetetramine or phosphotungstic acid molecules are immobilized by hydrogen-bonding with water molecules from above or from below this matrix, respectively. Thus, topochemical reactions between the epoxide oligomer and triethylenetetramine or between the epoxide oligomer molecules in the presence of phosphotungstic acid become possible, so that ultrathin network epoxide-triethylenetetramine or epoxide layers are formed on the surface of water.     

Prophylactic effects of ellagic acid and rosmarinic acid on doxorubicin‐induced neurotoxicity in rats

Doxorubicin (DOX) is a chemotherapeutic agent widely used in human malignancies. Its long‐term use cause neurobiological side effects. The aim of the present study was to investigate the prophylactic effect exerted by daily administration of ellagic acid (EA) and rosmarinic acid (RA) on DOX‐induced neurotoxicity in rats. Our data showed that DOX‐induced significant elevation of brain malondialdehyde, tumor necrosis factor‐alpha (TNF‐α), inducible nitric oxide synthase (iNOS), caspase‐3, and cholinesterase associated with significant reduction in reduced glutathione, monoamines namely serotonin, dopamine, as well as norepinephrine. Concomitant administration of EA (10 mg/kg/day, p.o. for 14 days) and/or RA (75 mg/kg/day, p.o. for 14 days) with DOX significantly mitigated the neural changes induced by DOX. Meanwhile, treatment ameliorated pro‐inflammatory cytokines as TNF‐α, iNOS, and attenuated oxidative stress biomarkers as well as brain monoamines. In conclusion, EA and RA can effectively protect against DOX‐induced neurotoxicity, and the mechanisms underlying the neuroprotective effect are potentially associated with its antioxidant, anti‐inflammatory, and antiapoptotic properties.     

Cyclophosphamide-induced nephrotoxicity, genotoxicity, and damage in kidney genomic DNA of Swiss albino mice: the protective effect of Ellagic acid

Cyclophosphamide (CPM), an alkylating agent is used as an immunosuppressant in rheumatoid arthritis and in the treatment of several cancers as well. In this study, Ellagic acid (EA), a naturally occurring plant polyphenol, was evaluated for its antigenotoxicity and antioxidant efficacy against the CPM-induced renal oxidative stress and genotoxicity in Swiss albino mice. The mice were given a prophylactic treatment of EA orally at a dose of 50 and 100 mg/kg body weight (b wt) for seven consecutive days before the administration of a single intraperitoneal (i.p.) injection of CPM at 50 mg/kg b wt. The modulatory effects of EA on CPM-induced nephrotoxicity and genotoxicity were investigated by assaying oxidative stress biomarkers, serum kidney toxicity markers, DNA fragmentation, alkaline unwinding assay, micronuclei (MN) assay, and by histopathological examination of kidney tissue. A single intraperitoneal administration of CPM in mice increased malondialdehyde level with depletion in glutathione content, antioxidant enzymes activities, viz. glutathione peroxidase, glutathione reductase, catalase, quinone reductase, induced DNA strand breaks, and MN induction. EA oral administration at both doses caused significant reduction in their levels, restoration in the activities of antioxidant enzymes, reduction in MN formation, and DNA fragmentation. Serum toxicity marker enzymes like BUN, creatinine, and LDH were also increased after CPM treatment which was significantly decreased in EA pretreated groups. Present findings suggest a prominent role of EA against CPM-induced renal injury, DNA damage, and genotoxicity.     

Ellagic acid protects dopamine neurons from rotenone‐induced neurotoxicity via activation of Nrf2 signalling

Parkinson's disease (PD) is the second most prevalent central nervous system (CNS) degenerative disease. Oxidative stress is one of key contributors to PD. Nuclear factor erythroid‐2‐related factor 2 (Nrf2) is considered to be a master regulator of many genes involved in anti‐oxidant stress to attenuate cell death. Therefore, activation of Nrf2 signalling provides an effective avenue to treat PD. Ellagic acid (EA), a natural polyphenolic contained in fruits and nuts, possesses amounts of pharmacological activities, such as anti‐oxidant stress and anti‐inflammation. Recent studies have confirmed EA could be used as a neuroprotective agent in neurodegenerative diseases. Here, mice subcutaneous injection of rotenone (ROT)‐induced DA neuronal damage was performed to investigate EA‐mediated neuroprotection. In addition, adult Nrf2 knockout mice and different cell cultures including MN9D‐enciched, MN9D‐BV‐2 and MN9D‐C6 cell co‐cultures were applied to explore the underlying mechanisms. Results demonstrated EA conferred neuroprotection against ROT‐induced DA neurotoxicity. Activation of Nrf2 signalling was involved in EA‐mediated DA neuroprotection, as evidenced by the following observations. First, EA activated Nrf2 signalling in ROT‐induced DA neuronal damage. Second, EA generated neuroprotection with the presence of astroglia and silence of Nrf2 in astroglia abolished EA‐mediated neuroprotection. Third, EA failed to produce DA neuroprotection in Nrf2 knockout mice. In conclusion, this study identified EA protected against DA neuronal loss via an Nrf2‐dependent manner.     

HISTOCHEMISTRY OF MYELIN—XII ANIONIC STAINING OF MYELIN BASIC PROTEINS FOR HISTOLOGY, ELECTROPHORESIS AND ELECTRON MICROSCOPY

Abstract— Phosphotungstic acid haematoxylin, trypan blue and amidoblack techniques have been developed as anionic dye methods for staining myelin basic proteins. All methods displayed central and peripheral nervous system myelin in histochemical prepa rations and stained brain basic proteins in electrophoretic polyacrylamide gels: phosphotungstic acid haematoxylin appeared to be the most selective of these techniques. Electron photomicrographs of peripheral nerve stained by phosphotungstic acid haematoxylin showed that the major part of myelin basic protein is located in the period dense line. The basic proteins stained by phosphotungstic acid haematoxylin showed an early loss in rat sciatic nerve undergoing Wallerian degeneration and had completely disappeared from the centre of 20 plaques of multiple sclerosis.     

Ultrastructure of the salt glands of the mangrove,Avicennia marina (Forssk.) Vierh., as indicated by the use of selective membrane staining

Each salt-excreting gland of the mangrove Avicennia marina (Forsskål) Vierh. consists of two to four collecting cells, one stalk cell, and eight to twelve excretory cells. Differential membrane staining by zinc iodide-osmium tetroxide (as a post-fixative) or phosphotungstic acid (as a section-stain) was used to characterise the ultrastructure of the glands. A large amount of tubular endoplasmic reticulum was found in the stalk and excretory cells of the gland, but not in the collecting cells. The ultrastructural arrangement of the endoplasmic reticulum indicates that salt is loaded from the apoplasm into the endoplasmic reticulum of the symplasm at the base of the stalk cell, traverses both cell types in the endoplasmic reticulum, and is excreted at the outer edge of the gland by an eccrine-type mechanism. Increasing development of the tubular endoplasmic reticulum accompanied differentiation of the gland cells.Abbreviations ER endoplasmic reticulum - PTA phosphotungstic acid - ZIO zinc iodide-osmium tetroxide     

Rhynchophylline attenuates migraine in trigeminal nucleus caudalis in nitroglycerin-induced rat model by inhibiting MAPK/NF-кB signaling

Cis-diamminedichloroplatinum(II) (cisplatin) (CP) is an important chemotherapeutic agent used in the treatment of several cancers. However, it has several side effects including nephrotoxicity gonadotoxicity, hepatotoxicity, and ototoxicity. In in vitro experiments, antioxidants or reactive oxygen species scavengers have a cytoprotective effect on cells exposed to cisplatin (CP). Ellagic acid (EA) is one such bioactive polyphenol that is abundant in some fruits, nuts, and seeds. Various authors have reported that EA has strong antioxidant and antitumor potential. The present study was, therefore, carried out to explore the protective potential of EA on CP-induced gonadotoxicity and nephrotoxicity in colon tumor-bearing mice. Animals were divided into five groups: Group I: normal control, Group II: DMH treated. After 20 weeks of DMH treatment, the animals were divided into four subgroups, viz., Group III: no treatment, Group IV: EA, Group V: CP, and Group VI: CP?+?EA. Administration of EA significantly ameliorated the toxicity caused by CP as indicated by improved kidney function tests and reproductive function tests. EA treatment to CP-abused mice also led to a marked reduction in the extent of peroxidative damage to tissue as was evident from the improvement in the histopathological changes in kidney and testis. Blood counts were also improved on administration of EA to CP-treated mice. This article provides the evidence that antioxidant efficacy of EA has beneficial effects on CP-induced nephrotoxicity and gonadotoxicity and contributes to understanding the role of oxidative stress, and suggests several points as part of the mechanism of CP toxicity.

     

Formation of 2-guanidinoethanol by a transamidination reaction from arginine and ethanolamine by the rat kidney and pancreas

The formation of 2-guanidinoethanol (GEt) from L-arginine (Arg) and ethanolamine (EA) was studied using rat kidney homogenates. Maximum GEt formation was observed between pH 8.7 and 9.1, and the enzyme catalyzing the GEt synthesis was stable between pH 5.6 and 9.1. The rate of GEt formation from Arg and EA by rat kidney homogenates obeyed simple Michaelis-Menten type kinetics. L-Ornithine and glycine inhibited GEt formation by rat kidneys. Both of them inhibited GEt formation in a linear mixed-type inhibitory manner when Arg concentrations were varied at a fixed concentration of EA, while they showed competitive inhibition when EA concentrations were varied at a fixed concentration of Arg. L-Canavanine and guanidinoacetic acid as well as Arg acted as an amidine donor for GEt formation, but L-homoarginine, 3-guanidinopropionic acid and 4-guanidinobutyric acid did not. GEt synthesis was also observed in the rat pancreas. It had almost half of the activity of rat kidney to form GEt. This ratio of kidney to pancreas was approximately equal to that of L-arginine:glycine amidinotransferase (transamidinase, EC 2.1.4.1) in kidney and pancreas. These results suggest that GEt may be synthesized from Arg and EA by a transamidinase catalyzing reaction.