A noninvasive mucinous cystic neoplasm with intermediate-grade dysplasia of the pancreas and extensive squamous metaplasia: a case report with clinicopathological correlation

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Metachromatic leukodystrophy (MLD) is a recessive autosomal disease which is characterized by an accumulation of sulfatides in the central and peripheral nervous system. It is due to the enzyme deficiency of the sulfatide sulfatase i.e. arylsulfatase A (ASA). we studied 5/200 cases of MLD and clearly distinguished three clinical forms. One of them presented the juvenile form; two presented the late infantile form; and two other presented the adult form. The Magnetic Resonance Imaging (MRI) of these patients showed a diffuse, bilateral and symmetrical demyelination. The biochemical diagnosis of MLD patients evidencing the low activity of ASA and sulfatide accumulation. Virtual slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1791578262610232

Patients and methods

We studied 5/200 MLD patients addressed to us for behavioral abnormalities and progressive mental deterioration. All of them were diagnosed at first by brain MRI evidencing a bilateral demyelination, then the measurement of ASA activity using P-nitrocathecol sulfate as substrate, finally the sulfatiduria was performed using thin-layer chromatography using alpha-naphtol reagent.

Results

In this study, from 200 patients presenting behavioral abnormalities and a progressive mental deterioration, we reported just 2 patients were diagnosed as late-infantile form of MLD. Only1 case presented as the juvenile form; and 2 patients with the adult-type of MLD. The brain magnetic resonance imaging (MRI) of all patients showed characteristic lesions of MLD with extensive demyelination. Biochemical investigations of these patients detected a low level of ASA activity at 0°C and 37°C; the excess of sulfatide in sulfatiduria.

Conclusion

MRI is required to orient the diagnosis of MLD patients; the latter must be confirmed by the biochemical investigations which is based on the measurement of ASA activity and the excess of sulfatide showed in the sulfatiduria.     

Deletion of fatty acid amide hydrolase reduces lyso-sulfatide levels but exacerbates metachromatic leukodystrophy in mice

An inherited deficiency of arylsulfatase A (ASA) causes the lysosomal storage disease metachromatic leukodystrophy (MLD) characterized by massive intralysosomal storage of the acidic glycosphingolipid sulfatide and progressive demyelination. Lyso-sulfatide, which differs from sulfatide by the lack of the N-linked fatty acid, also accumulates in MLD and is considered a key driver of pathology although its concentrations are far below sulfatide levels. However, the metabolic origin of lyso-sulfatide is unknown. We show here that ASA-deficient murine macrophages and microglial cells express an endo-N-deacylase that cleaves the N-linked fatty acid from sulfatide. An ASA-deficient astrocytoma cell line devoid of this activity was used to identify the enzyme by overexpressing 13 deacylases with potentially matching substrate specificities. Hydrolysis of sulfatide was detected only in cells overexpressing the enzyme fatty acid amide hydrolase (FAAH). A cell-free assay with recombinant FAAH confirmed the novel role of this enzyme in sulfatide hydrolysis. Consistent with the in vitro data, deletion of FAAH lowered lyso-sulfatide levels in a mouse model of MLD. Regardless of the established cytotoxicity of lyso-sulfatide and the anti-inflammatory effects of FAAH inhibition seen in mouse models of several neurological diseases, genetic inactivation of FAAH did not mitigate, but rather exacerbated the disease phenotype of MLD mice. This unexpected finding was reflected by worsening of rotarod performance, increase of anxiety-related exploratory activity, aggravation of peripheral neuropathy, and reduced life expectancy. Thus, we conclude that FAAH has a protective function in MLD and may represent a novel therapeutic target for treatment of this fatal condition.     

Compound heterozygosity for metachromatic leukodystrophy and arylsulfatase A pseudodeficiency alleles is not associated with progressive neurological disease

Several allelic mutations at the arylsulfatase A (ASA) locus cause substantial deficiencies of this lysosomal enzyme. Depending on the genetically determined degree of the deficiency, the clinical outcome may be very different—either metachromatic leukodystrophy (MLD), a lethal lysosomal storage disorder affecting the nervous system, or, more frequently, the so-called pseudodeficiency (PD), which has no apparent clinical consequence. Because of compound heterozygosity for MLD and PD, 1/1,000 individuals in the population have low residual enzyme activities, which are intermediate between those of MLD patients and those of PD homozygous normal individuals. In order to assess whether PD/MLD compound heterozygotes bear a health risk, we examined clinically and biochemically 16 individuals with this genotype. Of these subjects, two had neurological symptoms and two showed lesions, without clinical symptoms, in magnetic resonance imaging of the brain. None of these symptoms was progressive, nor did they resemble those of MLD. Nerve conduction velocities were normal in these probands, and they secreted only low amounts of sulfatide in the urine. We conclude that the observed neurological symptoms are unrelated to the ASA genotype and that PD/MLD compound heterozygotes are not at an increased risk for developing progressive nervous system diseases.     

Brain Galactolipid Content in a Patient with Pseudo-Arylsulfatase A Deficiency and Coincidental Diffuse Disseminated Sclerosis, and in Patients with Metachromatic, Adreno-, and Other Leukodystrophies

A 4-year old boy died of diffuse disseminated sclerosis (DDS) of the brain and was found to have also pseudoarylsulfatase A deficiency (PASAD) with about 20% residual arylsulfatase A (ASA) and cerebroside sulfatase (CS) activity. The reexamination of lipids did not show any sulfatide accumulation in the patient's organ extracts. Although the residual CS activity in the patient's extracts was clearly demonstrable only after partial purification, it was concluded that this activity protects organ tissues from sulfatide accumulation in PASAD, since in sulfatide lipidosis (metachromatic leukodystrophy, MLD) no residual CS activity was detectable. The study of residual ASA activity in the patient's fibroblasts by gel electrofocusing resulted in an almost normal enzyme microheterogeneity. However, the detailed study of the brain galactolipids in the patient revealed an elevated ratio of sulfatide/galactocerebroside content, despite the decrease of both lipids. In tissues of other patients with severe demyelinating diseases different from DDS and MLD, this galactolipid ratio was also found to be increased, especially in three patients with adrenoleukodystrophy. A general mechanism of this anomaly in severe demyelination is considered.     

Significance of the Leaf Area Ratio in Hevea brasiliensis Under High Irradiance and Low Temperature Stress

Adjustment in leaf area : mass ratio called leaf area ratio (LAR) is one of the strategies to optimize photon harvesting. LAR was recorded for 10 genotypes of Hevea brasiliensis under high irradiance and low temperature and the genotypes were categorized into two groups, i.e. high LAR and low LAR types. Simultaneously, the growth during summer as well as winter periods, photosynthetic characteristics, and in-vitro oxidative damage were studied. Low LAR (19.86±0.52 m² kg^–1) types, recorded an average of 18.0 % chlorophyll (Chl) degradation under high irradiance and 7.1 % Chl degradation under low temperature. These genotypes maintained significantly higher net photosynthetic rate (P _N) of 10.4 mol(CO₂) m^–2 s^–1 during winter season. On the contrary, the high LAR (24.33±0.27 m² kg^–1) types recorded significantly lower P _N of 4 mol(CO₂) m^–2 s^–1 and greater Chl degradation of 37.7 and 13.9 % under high irradiance and low temperature stress, respectively. Thus LAR may be one of the physiological traits, which are possibly involved in plant acclimation process under both stresses studied.     

Combined Effect of Irradiance and Water Regime on Sorghum Photosynthesis

Grain sorghum [Sorghum bicolor (L.) Moench. cvs. TX430 and KS82] was grown in a Haynie very fine sandy loam (coarse-silty, mixed, superactive, calcareous, mesic Mollic Udifluvents) under constant 47 % shade or full irradiance in a greenhouse under two watering regimes to see the combined and individual effects of low irradiance (LI) and low water (LW) on the sorghum genotypes. Under the high-irradiance (HI) and high-water (HW) treatment (control) and the LI-HW treatment, TX430 grew taller than KS82. Both LI and LW reduced several times the fresh and dry masses. Under the control conditions, TX430 reached its maximum net photosynthetic rate (P _Nmax) of 28.93 mol m^–2 s^–1 at a photosynthetic photon flux density (PPFD) of 1 707 mol m^–2 s^–1, and KS82 reached its P _Nmax of 28.32 mol m^–2 s^–1 at a PPFD of 2 973 mol m^–2 s^–1. The fact that TX430 had P _Nmax under a lower PPFD than KS82 may relate to its taller growth under LI conditions. Hence genotypes of sorghum might be selected for low irradiance using curves relating P _N to PPFD.     

A new polymorphism of arylsulfatase A within the coding region

A 10-year-old boy with juvenile metachromatic leukodystrophy (MLD) presented with the 459 + 1G→A arylsulfatase A (ASA) mutation on one allele. To detect his complete genotype, the other ASA allele was sequenced and a T-to-C transition at nucleotide 376 in exon 2 was identified. This missense mutation results in a substitution of leucine 76 by proline. Of 20 MLD unrelated controls, 18 carried the L/P76 mutation either in the homozygous (n = 6) or heterozygous (n = 12) state. The presence or absence of L/P76 did not influence leukocyte ASA activity or urinary sulfatide excretion. Apparently, the substitution of leucine 76 by proline is a common ASA polymorphism, neither being related to MLD nor creating ASA pseudodeficiency. However, because of its frequency and location, L/P76 may be of particular importance in genetic studies requiring the differentiation of the ASA alleles within a kindred. Further studies are directed to the as yet unresolved genotype of the index case with juvenile MLD. Received: 5 March 1996 / Revised: 16 April 1996     

Arylsulfatase A in pseudodeficiency

Summary Arylsulfatase A (ASA) is found to be deficient in healthy individuals (pseudo arylsulfatase A deficiency) who usually show in vitro ASA levels in the range of metachromatic leukodystrophy patients. The in vitro properties of ASA in pseudodeficiency were studied in cultured fibroblasts. The residual ASA activity showed apparent Km with the synthetic substrate (2.6mM), pH optimum of activity (pH 5.0), and sensitivity to heat denaturation at 65°C (T1/2, 10 min) similar to those found in controls. To test whether the low in vitro activity is a result of extreme sensitivity to the homogenization procedure, cells were disrupted by five different techniques, including rapid freezing and thawing, hand homogenization, ultrasonication, mild osmotic shock, and nitrogen cavitation; all yielded similar ASA ratio of the pseudodeficient to control. The use of antiproteases phenylmethylsulfonyl fluoride and leupeptin did not affect the residual ASA activity in the pseudodeficient line. These results imply that the ASA that is formed in this condition has properties similar to those of the normal hydrolase, so that even if it is synthesized in lower amounts, it is still sufficient to promote normal catabolism of sulfatide. Screening for ASA activity in lymphocyte extracts of a random sample of 250 individuals revealed 7 individuals with enzyme level in the MLD heterozygote range or lower. These individuals apparently represent homozygosity for pseudodeficiency (pd/pd). This implies that the frequency of the pseudodeficient allele is about 15% in the general population, leading to polymorphism of the ASA.     

Effects of elevated carbon dioxide on gas exchange and photochemical and nonphotochemical quenching at low temperature in tobacco plants varying in Rubisco activity

Elevated (700 μmol mol⁻¹) and ambient (350 μmol mol⁻¹) CO₂ effects on total ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity, photosynthesis (A), and photoinhibition during 6 d at low temperature were measured on wild type (WT), and rbcS antisense DNA mutants (T3) of tobacco (Nicotiana tabacum L.) with 60% of WT total Rubisco activity (Rodermel et al. (1988) Cell 55: 673–681). Prior to the low temperature treatment, A and quantum yield of PSII photochemistry in the light adapted state (φ_PSII) were significantly lower in T3 compared to WT at each CO₂ level. At this time, total nonphotochemical quenching (NPQ_Total) levels were near maximal (0.75–0.85) in T3 compared to WT (0.39–0.50). A was stimulated by 107% in T3 and 25% in WT at elevated compared to ambient CO₂. Pre-treatment acclimation to elevated CO₂ occurred in WT resulting in lower Rubisco activity per unit leaf area and reduced stimulation of A. At low temperature, A of WT was similar at elevated and ambient CO₂ while stimulation of A by elevated CO₂ in T3 was reduced. In addition, at low temperature we measured significantly lower photochemical quenching at elevated CO₂ compared to ambient CO₂ in both genotypes. NPQ_Total was similar (0.80–0.85) among all treatments. However, a larger proportion of NPQ_Total was composed of q_I,d, the damage subcomponent of the more slowly relaxing NPQ component, q_I, in both genotypes at elevated compared to ambient CO₂. Greater q_I,d, at elevated CO₂ during and after the low temperature treatment was not related to pre-treatment differences in total Rubisco activity.     

Effects of constitutive expression of nitrate reductase in transgenic Nicotiana plumbaginifolia L. in response to varying nitrogen supply

Transformed Nicotiana plumbaginifolia plants with constitutive expression of nitrate reductase (NR) activity were grown at different levels of nitrogen nutrition. The gradients in foliar NO ₃ ^– content and maximum extractable NR activity observed with leaf order on the shoot, from base to apex, were much decreased as a result of N-deficiency in both the transformed plants and wild type controls grown under identical conditions. Constitutive expression of NR did not influence the foliar protein and chlorophyll contents under any circumstances. A reciprocal relationship between the observed maximal extractable NR activity of the leaves and their NO ₃ ^– content was observed in plants grown in nitrogen replete conditions at low irradiance (170 mol photons·m^–2 ·s^–1). This relationship disappeared at higher irradiance (450 mol photons·m^–2·S^–1) because the maximal extractable NR activity in the leaves of the wild type plants in these conditions increased to a level that was similar to, or greater than that found in constitutive NR-expressors. Much more NO ₃ ^– accumulated in the leaves of plants grown at 450 mol photons·m^–2·s^–1 than in those grown at 170 mol photons·m^–2·s^–1 in N-replete conditions. The foliar NO ₃ ^– level and maximal NR activity decreased with the imposition of N-deficiency in all plant types such that after prolonged exposure to nitrogen depletion very little NO ₃ ^– was found in the leaves and NR activity had decreased to almost zero. The activity of NR decreased under conditions of nitrogen deficiency. This regulation is multifactoral since there is no regulation of NR gene expression by NO ₃ ^– in the constitutive NR-expressors. We conclude that the NR protein is specifically targetted for destruction under nitrogen deficiency. Consequently, constitutive expression of NR activity does not benefit the plant in terms of increased biomass production in conditions of limiting nitrogen.Abbreviations Chl chlorophyll - N nitrogen - NR NADH-nitrate reductase - WT wild type     

Effect of Salinity on Photosynthesis and Biochemical Characteristics in Mulberry Genotypes

Mulberry genotypes were subjected to salinity (0–12 mS cm^–1) in pot culture experiment. Chlorophyll and total carotenoid contents were reduced considerably by salinity. At low salinity, photosynthetic CO₂ uptake increased over the control, but it decreased at higher salinity. Contents of soluble proteins, free amino acids, soluble sugars, sucrose, starch, and phenols increased at salinity of 1–2 mS cm^–1 and decreased at higher salinity (8–12 mS cm^–1). Glycine betaine accumulated more than proline, the maximum accumulation of both was at salinity of 2–4 mS cm^–1. Among the genotypes studied, BC2-59 followed by S-30 showed better salinity tolerance than M-5.     

Response of Alfalfa Genotypes to Saline Water Irrigation

The influence of saline water (4, 8, 12 dS m^–1) irrigation on gas exchange and growth response of alfalfa genotypes Anand-2, T-9 and IL-112 was studied. T-9 and IL-112 showed a significant increase in net photosynthetic rate (P_N) at low salinity (4 dS m^–1) compared to the control whereas Anand-2 maintained an unaltered P_N. Reduction in P_N at higher salinities was primarily due to reduction of stomatal conductance. There was a greater reduction in transpiration rate as compared to P_N rate, which resulted in an increase in water use efficiency (WUE). High WUE may serve as one of the strategies of the plant to withstand saline environment. However, the slight increase in WUE in Anand-2 could not help in maintaining its growth. Increase in Na⁺ concentration in comparison to K⁺ concentration may also contribute to the inhibition in growth.     

Protective Effects of Exogenous Antioxidants and Phenolic Compounds on Photosynthesis of Wheat Leaves under High Irradiance and Oxidative Stress

Infiltration of methyl viologen (MV, source of O₂ ^–) and Na-diethyldithiocarbamate (DDC, inhibitor of SOD) into wheat leaves resulted in the accumulation of active oxygen species and photo-oxidative damage to photosynthetic apparatus under both moderate and high irradiance. Exogenous antioxidants, ascorbate (ASA) and mannitol, scavenged active oxygen efficiently, protected the photosynthetic system from MV and DDC induced oxidative damage, and maintained high F_v/F_m [maximal photochemical efficiency of photosystem 2 (PS2) while all PS2 reaction centres are open], F_m/F₀ (another expression for the maximal photochemical efficiency of PS2), _PS2 (actual quantum yield of PS2 under actinic irradiation), q_P (photochemical quenching coefficient), P _N (net photosynthetic rate), and lowered q_NP (non-photochemical quenching coefficient) of the leaves kept under high irradiance and oxidative stress. Phenolic compounds used in these experiments, catechol (Cat), resorcinol (Res), and tannic acid (Tan), had similar anti-oxidative activity and protective effect on photosynthetic apparatus as ASA and mannitol. The anti-oxidative activity and the protective effect of phenolic compounds increased with increase in their concentration from 100 to 300 g m^–3. The number and the position of hydroxyl group in phenolic molecules seemed to influence their antioxidative activity.     

Isolation of a highly active H+-ATPase from beef heart mitochondria

The lysolecithin extraction procedure originally described by Sadleret al. (1974) has been modified to yield a H⁺-ATPase with high levels of P_i-ATP exchange activity (400–600 nmol × min^–1 × mg^–1). This activity is further enhanced (1400–1600 nmol × min^–1 × mg^–1) following sucrose density gradient centrifugation in the presence of asolectin. This enhancement results in part from a lipid-dependent activation and in part from removal of inactive complexes. The H⁺ translocating activity of the complex has been determined spectrophotometrically using binding of oxonol VI as an indicator of membrane potential. P_i-ATP exchange, ATP hydrolysis, and oxonol binding are sensitive to energy-transfer inhibitors (oligomycin, rutamycin) and/or uncouplers (DNP, FCCP).     

Oxidative Stress and Taxol Production Induced by Fungal Elicitor in Cell Suspension Cultures of Taxus Chinensis

Treatment of Taxus chinensis cell suspension cultures with fungal elicitor resulted in an oxidative stress characterized by H₂O₂ production, malondiadehyde (MDA) accumulation and cell death. This oxidative stress was dependent on the concentration of elicitor. Cells exposed to elicitor accumulated taxol, however, not proportional to elicitor concentration. High production of taxol occurred in cells treated with the suitable elicitor concentration. We concluded that oxidative stress had the deleterious effect on taxol production. Simultaneous treatment with elicitor and ascorbic acid (ASA) changed the oxidative stress and taxol production. Production of taxol in cells treated with 200 mg dm^–3 elicitor and ASA was enhanced compared with that in cells treated with only 200 mg dm^–3 elicitor, while production of taxol in cells treated with 100 and 50 mg dm^–3 elicitor and ASA was decreased compared with that in cells treated with 100 and 50 mg dm^–3 elicitor.     

An adult-type metachromatic leukodystrophy caused by substitution of serine for glycine-122 in arylsulfatase A

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease with autosomal recessive inheritance caused by a deficiency of the enzyme arylsulfatase A (ASA). We have identified a new mutation in the ASA gene of a patient with adult-type MLD. In this mutation, the glycine at position 122, a highly conserved residue in the AS gene family, was replaced by serine. In a transient expression study, COS cells transfected with the mutant cDNA carrying ¹²²GlySer did not show an increase of ASA activity and produced little material immunoreactive to an anti-ASA antibody, despite normal mRNA levels.     

The relationship of nitrogen source and in vivo nitrate reductase actiity to root formation in Euphorbia esula cell suspension cultures

Root formation and in vivo nitrate reductase (NR) activity were determined in leafy spurge cell suspensions. Cells grown in B5 media with 1 mg L^–1 2,4-D were transferred to B5 media without 2,4-D, but containing either high (92:8) or low (15:85) ratios of nitrogen as NO ₃ ^– -N:NH ₄ ⁺ -N. In older cell lines root formation occurred only in the low NO ₃ ^– medium with =<30 roots per flask. In younger cell lines root numbers were greatest in the high NO ₃ ^– medium (1000 to 3000 per flask). Cells grown in low NO ₃ ^– medium were about one-third the final dry weight as those in high NO ₃ ^– medium. Root length was consistently greater for cell lines of all ages in the low NO ₃ ^– medium. Developmental profiles of NR activity were similar in cell lines of all ages, whether or not roots were formed. NR activity was lower, however, in cultures grown in low NO ₃ ^– medium compared to high NO ₃ ^– medium. There was no consistent relationship between NR activity and root initiation. Therefore, nitrate reductase does not appear to be a primary target for regulation of leafy spurge growth by chemical application.     

Photoinhibition and Active Oxygen Species Production in Detached Apple Leaves During Dehydration

In the course of dehydration, the gas exchange and chlorophyll (Chl) fluorescence were measured under irradiance of 800 mol m^–2 s^–1 in detached apple leaves, and the production of active oxygen species (AOS), hydrogen peroxide (H₂O₂), superoxide (O₂ ^–), hydroxyl radical (–OH), and singlet oxygen (¹O₂), were determined. Leaf net photosynthetic rate (P _N) was limited by stomatal and non-stomatal factors at slight (2–3 h dehydration) and moderate (4–5 h dehydration) water deficiency, respectively. Photoinhibition occurred after 3-h dehydration, which was defined by the decrease of photosystem 2 (PS2) non-cyclic electron transport (P-rate). After 2-h dehydration, an obvious rise in H₂O₂ production was found as a result of photorespiration rise. If photorespiration was inhibited by sodium bisulfite (NaHSO₃), the rate of post-irradiation transient increase in Chl fluorescence (Rfp) was enhanced in parallel with a slight decline in P-rate and with an increase in Mehler reaction. At 3-h dehydration, leaf P-rate decrease could be blocked by glycine (Gly) or methyl viologen (MV) pre-treatment, and MV was more effective than Gly at moderate drought time. AOS (H₂O₂ and O₂ ^–), prior to photoinhibition produced from photorespiration and Mehler reaction in detached apple leaves at slight water deficiency, were important in dissipating photon energy which was excess to the demand of CO₂ assimilation. So photoinhibition could be effectively prevented by the way of AOS production.     

Variability in nitrogen fixation of common bean (Phaseolus vulgaris L.) intercropped with maize

Thirty one selected bean lines were evaluated in the field for ability to support N₂ fixation when intercropped with maize which received 0, 30 and 60 kg N ha^–1 as ammonium sulphate. The amount of fixed N₂ was estimated using the natural variation of ¹⁵N and wheat as the standard non-fixing crop. Nitrogen as low as 15 kg N ha^–1 at sowing suppressed nodule weight and activity (acetylene reduction activity) but not nodule number, suggesting that the main effect of mineral N was on nodule development and function. ¹⁵N data revealed a high potential of the bean genotypes to fix N₂, with the most promising ones averaging between 50–60% of seed N coming from fixation. Bean lines CNF-480, Puebla-152, Mexico-309, Negro Argel, CNF-178, Venezuela-350 and WBR22-3, WBR22-50 and WBR22-55 were ranked as good fixers.     

Cl− channels in basolateral renal medullary membranes: VII. Characterization of the intracellular anion binding sites

A unique property of basolateral membrane Cl^– channels from the mTAL is that the Cl^– concentration facing the intracellular aspects of these channels is a determinant of channel open time probability (P ₀ ). The K _1/2 for maximal activation of P ₀ by Cl^– facing intracellular domains of these channels is 10 mm Cl^–. The present experiments evaluated the nature of these Cl^–-interactive sites. First, we found that the impermeant anion isethionate, when exposed to intracellular Cl^– channel faces, could augment P ₀ with a K _1/2 in the range of 10 mm isethionate without affecting conductance (g _Cl, pS). Second, pretreatment of the solutions facing the intracellular aspects of the channels with either 1 mm phenylglyoxal (PGO), an arginine-specific reagent, or the lysine/terminal amine reagent trinitrobenzene sulfonic acid (TNBS, 1 mm), prevented the activation of P ₀ usually seen when the Cl^– concentration of solutions facing intracellular channel domains was raised from 2 to 50 mm. However, when the Cl^– channel activity was increased by first raising the Cl^– concentration bathing intracellular channel faces from 2 to 50 mm, subsequent addition of either PGO or TNBS to solutions bathing intracellular Cl^– channel faces had no effect on P ₀ . We conclude that the intracellular aspects of these Cl^– channels contain Cl^–-interactive loci (termed [Cl] _i ) which are accessible to impermeant anions in intracellular fluids and which contain arginineand lysine-rich domains which can be inactivated, at low ambient Cl^– or isethionate concentrations, by interactions with PGO or TNBS.We acknoeledge the able technical assistance of Anna Grace Stewart. Clementine M. Whitman provided her customary excellent secretarial assistance. This work was supported by Veteterans Administration Merit Review Grants to T. E.Andreoli and to W. B. Reeves. C. J. Winters is a Veterans Administration Associate Investigator.