Mechanism for inhibition of thyroid peroxidase by leucomalachite green

The triphenylmethane dye, malachite green (MG), is used to treat and prevent fungal and parasitic infections in the aquaculture industry. It has been reported that the reduced metabolite of MG, leucomalachite green (LMG), accumulates in the tissues of fish treated with MG. MG is structurally related to other triphenylmethane dyes (e.g., gentian violet and pararosaniline) that are carcinogenic in the liver, thyroid, and other organs of experimental animals. The ability of LMG to inhibit thyroid peroxidase (TPO), the enzyme that catalyzes the iodination and coupling reactions required for thyroid hormone synthesis, was determined in this study. LMG inhibited TPO-catalyzed tyrosine iodination (half-maximal inhibition at ca. 10 microM). LMG also inhibited the TPO-catalyzed formation of thyroxine in low-iodine human goiter thyroglobulin (half-maximal inhibition at ca. 10 microM) using a model system that measures simultaneous iodination and coupling. Direct inhibition of the coupling reaction by LMG was shown using a coupling-only system containing chemically preiodinated thyroglobulin as the substrate. Incubation of LMG with TPO, iodide, and tyrosine in the presence of a H2O2-generating system yielded oxidation products that were identified by using on-line LC/APCI-MS as desmethyl LMG, 2desmethyl LMG, 3desmethyl LMG, MG, and MG N-oxide. Similar products from LMG were observed in incubations with TPO and H2O2 alone. These findings suggest that the anti-thyroid effects (increased serum thyroid-stimulating hormone and decreased serum thyroxine) observed in rats treated with LMG result from blockade of hormone synthesis through alternate substrate inhibition and that chronic exposure could cause thyroid follicular cell tumors through a hormonal mechanism. The observed TPO-catalyzed oxidative demethylation of LMG to a primary arylamine also suggests a genotoxic mechanism for tumor formation is possible.     

Confirmation of malachite green, gentian violet and their leuco analogs in catfish and trout tissue by high-performance liquid chromatography utilizing electrochemistry with ultraviolet-visible diode array detection and fluorescence detection

A sensitive analytical procedure for the confirmation of residues of malachite green (MG), gentian violet (GV) and their leuco analogs (LMG and LGV) in catfish and trout tissue at 10 ng/g is described. Frozen (-20 degrees C) fish fillets were cut into small pieces and homogenized in Waring blendors. The compounds of interest were extracted from 20-g amounts of homogenized fish tissue with acetonitrile-buffer, partitioned against methylene chloride, and isolated with tandem neutral alumina and propylsulfonic acid cation-exchange solid-phase extraction cartridges. Samples of 100 microl (0.8 g equiv.) were chromatographed isocratically in 10 min using an acetonitrile-buffer mobile phase on a short-chain deactivated (SCD) reversed-phase column (150x4.6 mm I.D.) in-line with a post-column oxidation coulometric electrochemical cell (EC), a UV-Vis diode array detector and a fluorescence detector.     

T-cell receptors in channel catfish: structure and expression of TCR alpha and beta genes

Herein are reported full length cDNA sequences for TCR alpha- and beta-chains of the channel catfish. Included are sequences belonging to four Valpha and six Vbeta families which share hallmarks in common with the Valpha and Vbeta genes of other species. Similar to the situation in other vertebrates, the catfish Calpha and Cbeta sequences exhibit distinct immunoglobulin, connecting peptide, transmembrane and cytoplasmic domains. However, the catfish TCR Calpha and Cbeta regions are shorter than those of mammals and the catfish Cbeta chain lacks a cysteine in its connecting peptide region. Two different catfish Cbeta cDNA sequences were identified, suggesting the existence of either two Cbeta loci or allotypes. Based on Southern blot analyses, each of the catfish TCR gene loci appear to be arranged in a translocon (as opposed to multicluster) organization with multiple V elements and a single or few copies of C region DNA. At the deduced amino acid level, the catfish Cbeta sequence exhibits 42% identity with the Cbeta of Atlantic salmon, 41% identity with the Cbeta of rainbow trout and 26% identity with Cbeta of the horned shark. The catfish Calpha amino acid sequence exhibits 44 and 29% identity with Calpha of the rainbow trout and southern pufferfish, respectively. TCRalpha and beta messages are selectively expressed and rearranged in a catfish clonal cell line that appears to be of the T lineage. This TCR alpha/beta expressing clonal lymphocyte line, designated 28S.1, has T-cell like function in that it constitutively produces a supernatant factor(s) with growth promoting activity. These findings should facilitate functional studies of fish TCRs and T cells in ways not previously possible with other 'lower' vertebrate models.     

Prominent increase of macrophage migration inhibitory factor in the sera of patients with uveitis

Ciguatera is a significant food-borne disease caused by potent polyether toxins (ciguatoxins) which accumulate in the flesh of ciguateric reef fish at risk levels > 0.1 ppb for Pacific ciguatoxins. Research on ciguatera has been severely hindered by the lack of analytical methods that detect and characterize low levels of ciguatoxin in crude extracts of fish. Here we report a new procedure for ciguatoxin analysis based on gradient reversed-phase HPLC/tandem mass spectrometry (HPLC/MS/MS). The method gave a linear response to pure Pacific and Caribbean ciguatoxins (P-CTX-1 and C-CTX-1) and the structurally related brevetoxin (PbTx-2) spiked into crude extracts of fish. Levels equivalent to 40 ppt P-CTX-1, 100 ppt C-CTX-1, and 200 ppt PbTx-2 in fish flesh could be detected by HPLC/MS/MS. Using P-CTX-1 as an internal standard, the analysis of extracts of 30 ciguateric fish from the Caribbean Sea (8 toxic, 12 borderline, and 10 nontoxic by mouse bioassay) confirmed the reliability of the method and allowed an estimated risk level of > 0.25 ppb C-CTX-1 to be established. HPLC/MS/MS provides a sensitive analytical approach, not previously available, for the determination of Pacific and Caribbean ciguatoxins at sub-ppb levels in fish flesh.     

Immunochemical characterization of hepatic cytochrome P450 isozymes in the channel catfish: assessment of sexual, developmental and treatment-related effects

The profiles of immunoreactive proteins recognized by antibodies raised against purified trout P-450 isoforms (CYP1A1, CYP2M1 and CYP2K1) were examined in channel catfish liver by Western blot analysis. Gender differences in basal expression of these isoforms, as well as responses to known inducers of mammalian isoforms (ethanol, beta-naphthoflavone and clofibric acid) and early life stage (3 and 6 months) profiles are described. Two similar protein bands were detected by Western blotting in mature untreated catfish with CYP2K1 and CYP2M1 antibodies. A third band is detected by anti-2K1 in fish treated with beta-naphthoflavone; this band was verified as CYP1A, with about twice the level of expression in males versus females. No difference between sexes was seen in the expression of the 51-kDa CYP2-reactive bands; however, a significant difference (female > male) was seen in the lower molecular weight CYP2 band (47-kDa). Ethanol treatment caused a dose-dependent decrease in the 47-kDa CYP2-reactive isoforms but no change in the 51-kDa band. Clofibric acid treatment caused an increase in both the 51-kDa CYP2 protein as well as in liver somatic index. Age-dependent changes in isoform expression were also detected in CYP2-reactive forms, with a novel protein (53-kDa) detected in 3-month-old fish. The results from this study provide insight into the regulation of constitutive catfish CYP isoforms and prepares a foundation for further examination of the biotransformation capabilities of an important aquatic species.     

Comparative studies of suidatrestin, a specific inhibitor of trehalases

The ability to synthesise prostaglandins and thromboxane from 14C-labelled arachidonic acid was investigated in 11 species of fish from the Arabian Gulf. Cyclooxygenase activity was assessed in washed whole blood cells. Arachidonic acid and its metabolites were extracted and separated on silicic acid columns and thin layer chromatography (silica gel G). Total capacity to convert [14C]arachidonic acid to prostanoids varied from 1 to 35% among the 11 fish species studied. Gray shark (Chiloscyllium griseum) blood cells had the highest capacity (37 +/- 0.4%) to convert arachidonate into prostanoids and two species of catfish (Arius bilineatus and A. thalassinus) exhibited greater than 10% capacity to convert [14C]arachidonate into prostanoids. The major prostanoid synthesised by the two catfish (A. bilineatus and A thalassinus) was 6-keto PGF1 alpha, a stable metabolite of prostacyclin, PGI2. In contrast, A. teunispinis synthesised thromboxane B2, a stable metabolite of thromboxane A2. Thromboxane B2 (TXB2) was the major product synthesised by all three species of shark studied (Chil. griseum, Carcharhinus plumbeus, Carch. melanopterus), with 6-keto PGF1 alpha a minor product. Other fish studied showed a varied pattern of prostanoid synthesis. The synthesis of these prostanoids was almost completely blocked by preincubation of the whole blood cells from catfish and shark with indomethacin (0.5 microM) suggesting the involvement of cyclooxygenase-mediated prostanoid synthesis.     

Determination of sulfadimethoxine withdrawal time from milk. Part II: Statistical assessment

A statistical method is proposed to establish milk discard time for the data set described in Part I (preceding paper). Results are compared with those from the Food and Drug Administration (FDA)-recommended method. The milk discard time is established on the basis of a calculated tolerance limit. This limit provides 95% confidence that 99% of the population residue would assay below the permitted concentration (10 ppb for SDM). Unlike the FDA method, the proposed method allows easy calculation and requires no assumptions in drug depletion rate over time. For a permitted concentration of 10 ppb, both methods confirm the present 60-h discard time for SDM when it is assumed that no more than 1/3 of the milk came from treated cows.     

Total radioactive residues and clenbuterol residues in edible tissues, and the stereochemical composition of clenbuterol in livers of broilers after exposure to three levels of dietary [14C]clenbuterol HCl and three preslaughter withdrawal periods

Thirty-six broiler chickens were randomly assigned to .5, 1.0, or 2.0 ppm dietary [14C]clenbuterol HCl for a 2-wk period starting at 5 wk of age. Four birds from each treatment were slaughtered after withdrawal periods of 0, 7, or 14 d. Total radioactive residues (TRR; clenbuterol HCl equivalents) were measured in adipose tissue, kidney, liver, skin with adhering adipose tissue, bile, blood, brain, gastrointestinal tract, heart, lung, spleen, and testes; parent clenbuterol was measured in liver and kidney. In edible tissues, TRR were roughly proportional to dietary [14C]clenbuterol level and inversely proportional to duration of the withdrawal period; kidney TRR ranged from nondetectable (14 d of withdrawal, .5 and 1.0 ppm treatments) to 211.5 ppb for the 2.0 ppm treatment at zero withdrawal. Liver TRR were detectable for all treatment and withdrawal periods. Rapid depletion of TRR from edible tissues occurred during the first 7 d of the withdrawal period, but depletion of TRR was much slower thereafter. Parent clenbuterol was below the limit of detection (1 ppb) or was undetectable in liver and kidney for all dietary levels after 7 and 14 d of withdrawal, but it represented 22 to 48% of the total radioactive residues at 0 withdrawal. The inactive S (+) stereoisomer constituted approximately 73% of the total clenbuterol residue in livers of chickens slaughtered with no withdrawal period, and the active R (-) stereoisomer accounted for the remainder. These data indicate that radioactive residues of clenbuterol were present well after parent clenbuterol had depleted from edible tissues in chickens, and the predominant stereoisomer remaining in livers at slaughter was the inactive isomer.     

Accumulation and elimination of dieldrin in muscle tissue of channel catfish

Dieldrin accumulation and elimination in muscle tissue of channel catfish (Ictalurus punctatus) from water and food was determined in the laboratory. Twenty-eight-day exposure of fish 150-225 mm and 350-400 mm long to 75 parts per trillion (ng/liter) dieldrin resulted in the larger catfish consistently accumulating more dieldrin than the smaller fish. After 28 days of elimination, dieldrin levels in both size groups were nearly equal. Catfish exposed to 2 ppm (mg/kg) dieldrin through their diets accumulated significantly more dieldrin in muscle than did fish exposed to 75 pptr in water. When fish were exposed to dieldrin both in food and water, dieldrin from both sources contributed to the total dieldrin load. Large catfish accumulated more dieldrin from food and water than did smaller catfish. After 28 days of elimination, levels of dieldrin were not significantly different in muscles of 150-225 mm and 350-440 mm catfish exposed via both food and water.     

On classification of changes in necrotizing lesions of the midface

Lethal midline granuloma syndrome (LMG) describes lesions of the midface and is characterized by a progressive and often fatal ulceration and destruction of the upper air way involving the nose, the paranasal sinuses, the palate and the soft tissues of the face. Under the term LMG we distinguish four entities: idiopathic midline destructive disease (IMDD), polymorphic reticulosis (PR), non Hodkin's lymphoma and Wegener's granulomatosis (WG). Review of the literature allows to present the differential diagnosis making use of latest technological achievements in clinical immunology and immunohistochemistry. First of all the LMG must be discriminated from localized WG occurring in the midface. The clinical, serological and histopathological findings in WG are described. Literature review is carried out and recent concepts of it's etiology and pathogenesis are presented. Autoantibodies directed against cytoplasmic antigens of neutrophils (ANCA) with specificity for proteinase 3 (PR 3) are valuable marker for differential diagnosis and specificity are discussed. We make a comparison between the "limited" or "non renal" WG and "classical" or "renal" form of the disease.     

Occupational asthma caused by fish inhalation

Occupational asthma (OA) due to fish inhalation, confirmed by specific bronchial challenge (SBC), has not been described as yet in medical literature, as far as we know. We describe two patients whose asthma was induced by occupational exposure to fish and confirmed by serial measurements of PEFR and SBC. Two fish-processing workers reported asthma symptoms related to their workplace. They were skin tested with fish extracts and their sera assayed for IgE antibodies to various fish species. Nonspecific bronchial reactivity was assessed by methacholine challenge. The occupational relationship was confirmed by PEFR monitoring in working and off-work periods. SBC with fish extracts was carried out to confirm the diagnosis of OA. Skin tests with raw and cooked plaice, salmon, hake, and tuna in patient 1 and anchovy, sardine, trout, salmon, Atlantic pomfret, and sole in patient 2 were positive. Specific IgE serum antibodies were found to salmon in patient 1 and to trout, anchovy, and salmon in patient 2. PEFR measurements differed significantly (P < 0.001) between work and off-work periods for both patients. A bronchial challenge with methacholine was positive in patient 1. SBC with raw hake, salmon, plaice, and tuna extracts in patient 1 and raw salmon extract in patient 2 were all positive with an immediate response. SBC with Dermatophagoides pteronyssinus extract was entirely negative in both patients. In three asthmatic, non-fish-allergic controls, SBC with tuna, hake, salmon, and plaice were all negative. These results suggest that fish inhalation can elicit IgE-mediated occupational asthma.     

Mercury levels in freshwater fish of the state of South Carolina

Samples of fish from freshwater sources of rivers, lakes and ponds all over the state of South Carolina were collected during the Summer of 1974 and 1975. The fish collected were Bass, Bluegill, Redbreast, Catfish, Shad, Carp, Crappie, Mudfish and Pike. Samples were analyzed using the flameless atomic absorption procedure outlined by Hatch and Ott, and Uthe et al as modified for use with Perkin-Elmer, Coleman MAS-50 mercury analyzer. Triplicate samples of fish tissue were analyzed by wet digestion method. The mean mercury levels in ppb were determined for baseline mercury levels. A significant finding of this report is that those species for which fish of widely differing weights were analyzed, larger fish had higher mercury levels. Mercury levels exceeding the U.S. Food and Drug Administration guideline of 500 ppb for fish tissues have been found in the Mudfish from Edisto River and Pike fish from Lake Murray. Higher levels of mercury occurred in the highly vascularized blood tissues of liver and kidney than in muscle. Carnivorous and bottom-feeding fishes are the most reliable indicators of mercury pollution.     

The problem of asbestos today

In the past 2 years, a 4 year-old boy has had an anaphylactic reaction whenever he contacted food prepared with fish. The symptoms included intense itching in the throat and eyes, which progressed to generalized urticaria and facial angioedema. This was accompanied by cough, wheezing and dyspnea. Many fish preparations caused these episodes including several different kinds of fish (cod, tuna, salmon, trout, eel...), fish soup, chopsticks contaminated with fish preparations and canned fish. Elevated levels of total serum IgE (224 IU/ml) and specific IgE for cod (93.1 IU/ml), tuna (> 100 IU/ml), salmon (> 100 IU/ml), trout (64.4 IU/ml), mackerel (41.2 IU/ml) and eel (28.1 IU/ml) were found by the Pharmacia CAP system RAST FEIA in our allergy clinic. A skin prick test for mixed fish extracts (contain flounder, cod and halibut) was positive. A fish challenge test for cod, tuna, salmon, trout and eel all showed anaphylactic reactions. His allergic symptoms stabilized gradually after strictly avoiding ingestion of fish and using drug treatment. He also had a similar anaphylactic reaction to frogs. The best treatment for fish allergy is avoidance. Avoidance of fish may need to include both ingestion and inhalation of cooking vapors.     

Determination of four anabolic steroid metabolites by gas chromatography/mass spectrometry with negative ion chemical ionization and tandem mass spectrometry

A gas chromatography/mass spectrometry method is described which uses negative ion chemical ionization and tandem mass spectrometry for the determination of anabolic steroid metabolites. Four anabolic steroid metabolites to be derivatized by Pentafluoropropionic anhydride (PFPA) were determined using gas chromatography/mass spectrometry (GC/MS) with negative chemical ionization (NCI) and NCI/MS/MS. The repeatability and reproducibility of this procedure were in the range of 5.3-9.7% and 6.1-10.2%, respectively. This method of derivatization with PFPA for NCI and NCI/MS/MS was useful to determine four metabolites of nandrolone, dromostanolone, methenolone and boldenone. The derivatized metabolites of boldenone could be detected to 2 ppb and the other three steroids could be detected to 25 ppb in urine at a signal-to-noise ratio of S/N = 3.     

Organochlorine pesticides and polychlorinated biphenyls (PCBs) in sediments and biota from four US Arctic lakes

Organochlorine (OC) concentrations in surface sediment, snails (Lymnea sp.), and two freshwater fish species (grayling, Thymallus arcticus; and lake trout, Salvelinus namaycush) from four lakes in the US Arctic were determined. In surface sediment, chlorinated benzenes (including hexachlorobenzene, HCB), and p,p'-DDT were the primary analytes detected (max = 0.7 ng/g dry wt), while individual polychlorinated biphenyl (PCB) congeners were always below 0.1 ng/g. A wider range of compounds and higher concentrations were found in lake trout, the top predatory fish species in the same lakes. The concentration ranges for hexachlorocyclohexanes (HCHs), chlordane-related compounds (CHLORs), DDTs, and PCBs in lake trout and grayling were similar to those reported for other arctic freshwater fish (1-100 ng/g wet wt), but one to two orders of magnitude lower than Great Lakes salmonids. Nitrogen isotope analysis confirmed that differences in OC concentrations between grayling and lake trout are explained partly by differences in food web position.     

750 MHz 1H NMR spectroscopy characterisation of the complex metabolic pattern of urine from patients with inborn errors of metabolism: 2-hydroxyglutaric aciduria and maple syrup urine disease

750 MHz 1H NMR spectroscopy has been used to characterise in detail the abnormal low molecular weight metabolites of urine from two patients with inborn errors of metabolism. One case of the rare condition 2-hydroxyglutaric aciduria has been examined. There is at present no rapid routine method to detect this genetic defect, although NMR spectroscopy of urine is shown to provide a distinctive pattern of resonances. Assignment of a number of prominent urinary metabolites not normally seen in control urine could be made on the basis of their known NMR spectral parameters including the diagnostic marker 2-hydroxyglutaric acid, which served to confirm the condition. In addition, 750 MHz 1H NMR spectroscopy has been used to characterise further the abnormal metabolic profile of urine from a patient with maple syrup urine disease. This abnormality arises from a defect in branched chain keto-acid decarboxylase activity and results in a build up in the urine of high levels of branched chain oxo- and hydroxy-acids resulting from altered metabolism of the branched chain amino acids, valine, leucine and isoleucine. A number of previously undetected abnormal metabolites have been identified through the use of one-dimensional and two-dimensional J-resolved and COSY 750 MHz 1H NMR spectroscopy, including ethanol, 2-hydroxy-isovalerate, 2,3-dihydroxy-valerate, 2-oxo-3-methyl-n-valerate and 2-oxo-isocaproate. NMR spectroscopy of urine, particularly when combined with automatic data reduction and computer pattern recognition using a combination of biochemical markers, promises to provide an efficient alternative to other techniques for the diagnosis of inborn errors of metabolism.     

Methionine requirement of channel catfish fed soybean meal-corn-based diets

A soybean meal-corn-based diet was used to determine dietary methionine (Met) required by 14-g channel catfish (Ictalurus punctatus) in a 42-d experiment at 25 degrees C. The basal diet with balanced limiting amino acids relative to the catfish whole-body amino acid profile contained 277 g of CP, 3.6 g of Met, 4.0 g of cystine (Cys), and 10 MJ of DE/kg of DM. DL-Methionine was added to the basal diet from 0 to 12.0 g/kg in 2-g intervals at the expense of L-glutamic acid to produce seven isonitrogenous and isocaloric diets. A reference diet contained 331 g of CP, 8 g of Met, 5 g of Cys, and 10 MJ of DE/kg of DM (included 8% fish meal). Seven graded Met levels resulted in quadratic responses (P < .01) of weight gain, specific growth rate, feed or GE intake, feed or energy efficiency, protein or energy retention, protein efficiency ratio, and apparent net protein or energy utilization. Channel catfish required 9.4 g of Met/kg of DM (34.1 g/kg of CP) with a total 11.3 g/kg of calculated digestible sulfur-containing amino acids based on multiple regression dose-response models or 270 mg of Met/kg of fish per day based on a broken-line response of protein gain to Met intake. At the adequate Met level, catfish with the lowest (P < .05) liver lipids showed feed intake and protein or energy utilization efficiency similar (P > .05) to that of catfish fed the reference diet. Catfish fed all-plant-protein diets require more dietary methionine than previously reported. Catfish fed corn-soybean meal diets fortified adequately with methionine result in performance that approaches that of fish fed a fish meal-based diet.     

Immunohistochemical detection of vitellogenin in male brown trout from Swiss rivers

The detection of vitellogenin, a yolk precursor protein, may serve as a biomarker for exposure to environmental oestrogens as its induction by xenobiotic oestrogens in the immature and male fish has been reported repeatedly. In the present work, juvenile brown trout were injected with oestradiol (5 microg g(-1) body weight oestradiol benzoate) in order to assess the induction and organ distribution of vitellogenin by means of immunohistochemistry. In addition, brown trout collected from Swiss rivers were analysed. Vitellogenin was detected in the oestradiol-injected juvenile trout but not in uninjected controls. The presence of vitellogenin was also demonstrated in a male and an immature feral brown trout from one of two locations downstream of three sewage treatment plants. In contrast, no positive staining was found in livers of trout upstream of the respective plants. The results demonstrate the suitability of immunohistochemistry for monitoring feral fish for the presence of vitellogenin production.     

Molecular basis for prokaryotic specificity of magainin-induced lysis

Magainins and mastoparans are examples of peptide antibiotics and peptide venoms, respectively. They have been grouped together as class L amphipathic helixes [Segrest, J.P., et al. (1990) Proteins 8, 103-117] because of similarities in the distribution of Lys residues along the polar face of the helix. Class L venoms lyse both eukaryotic and prokaryotic cells whereas class L antibiotics specifically lyse bacteria. The structural basis for the specificity of class L antibiotics is not well understood. Sequence analysis showed that class L antibiotics have a Glu residue on the nonpolar face of the amphipathic helix; this is absent from class L venoms. We synthesized three model class L peptides with or without Glu on the nonpolar face: 18LMG (LGSIWKFIKAFVGGIKKF), [E14]18LMG and [G5,E14]18LMG. Hemolysis, bacteriolysis, and bacteriostasis studies using these peptides showed that the specificity of lysis is due to both the presence of a Glu residue on the nonpolar face of the helix and the bulk of the nonpolar face. Studies using large unilamellar phospholipid vesicles showed that the inclusion of cholesterol greatly inhibited leakage by the two Glu-containing peptides. These results cannot be attributed to changes in the phase behavior of the lipids caused by the inclusion of cholesterol or to differences in the secondary structure of the peptides. These results suggest that eukaryotic cells are resistant to lysis by magainins because of peptide-cholesterol interactions in their membranes that inhibit the formation of peptide structures capable of lysis, perhaps by hydrogen bonding between Glu and cholesterol. Bacterial membranes, lacking cholesterol, are susceptible to lysis by magainins.     

The ARG11 gene of Saccharomyces cerevisiae encodes a mitochondrial integral membrane protein required for arginine biosynthesis

Prototype strain MG409 (arg11-1) is a severe arginine bradytroph with greatly reduced ornithine and arginine pools, although all known enzymes required for arginine biosynthesis are functional. To identify the function required for normal arginine production impaired in MG409, we have cloned, sequenced, and performed a first molecular characterization of ARG11. We show that the ARG11 open reading frame encodes a putative 292-residue protein with a predicted molecular mass of 31.5 kDa. Sequence similarities, a tripartite organization, and six potential hydrophobic transmembrane spans suggest that Arg11p belongs to the mitochondrial integral inner membrane carrier family. We have used immuno-Western blotting and hemagglutinin epitope-tagged derivatives of Arg11p, Arg8p (a mitochondrial matrix marker), and Arg3p (a cytosolic marker) to demonstrate that Arg11p is confined to the mitochondria and behaves like an integral membrane protein. A deletion created in ARG11 causes the same arginine-leaky behavior as the original arg11-1 mutation, which yields a premature stop codon at residue 266. Arg11p thus appears to fulfill a partially redundant function requiring its 27 carboxyl-terminal amino acids. As a working hypothesis, we propose that Arg11p participates in the export of matrix-made ornithine into the cytosol.