Coding of consonant-vowel transition in children with central auditory processing disorder: an electrophysiological study.

INTRODUCTION: Acoustic change complex (ACC) is an important tool to investigate the encoding of the acoustic property of speech signals in various populations. However, there is a limited number of research papers that have explored the usefulness of ACC as a tool to study the neural encoding of consonant-vowel (CV) transition in children with central auditory processing disorder (CAPD). Thus, the present study aims to investigate the utility of ACC as an objective tool to study the neural representation of consonant-vowel (CV) transition in children with CAPD. METHODS: Twenty children diagnosed having CAPD and 20 normal counterparts in the age range of 8-14 years were the participants. The ACC was acquired using naturally produced CV syllable /sa/ with a duration of 380 ms. RESULTS: Latency of N1' and P2' was found to be prolonged in children with CAPD compared to normal counterparts, whereas the amplitude of N1' and P2' did not show any significant difference. Scalp topography showed significantly different activation patterns for children with and without CAPD. CONCLUSION: Prolonged latencies of ACC indicated poor encoding of CV transition in children with CAPD. The difference in scalp topography might be because of the involvement of additional brain areas for the neural discrimination task in children with CAPD.

Effect of pre-transition stimulus duration on acoustic change complex.

OBJECTIVE: To investigate the effect of pre-transition duration on acoustic change complex (ACC) for speech and tonal stimulus. DESIGN: Cortical potentials were recorded for consonant-vowel syllable and tonal complex stimuli with varying pre-transition durations. STUDY SAMPLE: Ten individuals (three male and seven female) in the age range from 18 to 26 years, with normal hearing sensitivity, participated in the study. RESULTS: The results revealed that a minimum pre-transition duration of 100 ms for tonal stimulus (with spectral change) and 80 ms for consonant-vowel syllable is necessary in order to elicit ACC. The latency of N1(1) and P2(1), which is the response for change within the ongoing stimulus, increased with increase in pre-transition duration. The amplitude of the evoked responses did not show any significant change. CONCLUSIONS: It was found that minimum pre-transition duration required in eliciting ACC for speech and non-speech stimulus is not same. The speech stimulus required lesser duration of pre-transition than non-speech stimulus. Further studies regarding the acoustic aspects of sound on CAEP in isolation are warranted.

Diagnostic utility of serologic markers for genital chlamydial infection in STD patients in Chennai, India.

OBJECTIVES: To evaluate the diagnostic utility of serological markers for C. trachomatis in different clinical groups of STD patients. METHODS: Blood and genital swab specimens were collected from symptomatic STD patients (n=143) attending the STD out patient clinic at the Institute of STDs, Government General hospital, Chennai who enrolled for the study. Serological determination for IgM, IgA and IgG antibodies to C. trachomatis was done using commercial kits. PCR analysis was performed on genital swab samples by using plasmid and major outer membrane protein (MOMP) based primers and patients who were positive by both PCR assays were considered as proven cases of C. trachomatis infection. The serological marker positivity was analysed with PCR positivity. RESULTS: Serologic positivity by IgM, IgA and IgG was 22.4%, 28.7% and 58.7% respectively. The PCR analysis showed 44 (30.8%) cases with confirmed C. trachomatis infection. Seropositivity for IgM (34.1% (15/44) vs. 17.2% (17/99); P<0.05) as well as for IgA (40.9% (18/44) vs. 23.2% (23/99); P<0.05) significantly correlated to PCR positivity, while significant correlation was not seen with IgG positivity. The overall seropositivity (IgM/IgA/IgG) in the study population was 68.5%. CONCLUSIONS: The observations of the present study indicate a high exposure rate to chlamydial infection in STD clinic patients in India. The study also suggests the usefulness of serology instead of PCR to trace chlamydial etiology, especially in deep-seated upper genital tract diseases and to facilitate better clinical management as there was good correlation between serology and PCR positivity.

Amino Acid Transporter ATB0,+ as a delivery system for drugs and prodrugs.

ATB(0,+) is a unique amino acid transporter because of its broad substrate specificity and concentrative ability. This transporter recognizes neutral as well as cationic amino acids. It is energized by Na(+) and Cl(-) gradients and membrane potential. Many of the amino acids and amino acid derivatives that are substrates for ATB(0,+) serve as therapeutic agents (e.g., D-serine, carnitine, and nitric oxide synthase inhibitors). Recent studies have shown that the potential of ATB(0,+) as a drug delivery system may be greater than previously envisaged. ATB(0,+) can transport antiviral drugs such as acyclovir and ganciclovir when they are covalently coupled to the side chain of anionic amino acids. Chemical modification of the carboxyl groups in the side chain of aspartate and glutamate with drugs converts these anionic amino acids into neutral amino acid derivatives. Therefore, the modified drugs are recognized by ATB(0,+). Interestingly, even when acyclovir and ganciclovir are coupled as esters with alpha-carboxyl group of neutral amino acids, the modified drugs are transported via ATB(0,+). Similarly, the hydroxyl group in the side chains of serine and threonine can also be used to covalently couple drugs for delivery into cells via ATB(0,+). This increases the potential for designing a wide variety of amino acid-based prodrugs that can utilize ATB(0,+) as drug delivery system. Furthermore, the transporter is expressed in the colon, lung, and eye, the tissues easily amenable for drug delivery. These findings argue strongly in support of ATB(0,+) as a potential delivery system for a wide variety of drugs and prodrugs.

Genital chlamydial infection in STD patients: its relation to HIV infection.

In the present report, we have analysed C.trachomatis infection and HIV positivity among patients (n-143) who attended the STD clinic at the Institute of STDs, Government General Hospital, Chennai. HIV positivity rate was significantly high among those with chlamydial infection than in those without chlamydial infection (29.5% (13/44) vs. 11.1% (11/99); p<0.05). The results of the present study suggest the association between C.trachomatis and HIV infections and reinforce the need for routine screening for C.trachomatis as a necessary intervention to reduce the burden of chlamydial diseases and to reduce the risk of HIV and its spread in India.

Need for specific & routine strategy for the diagnosis of genital chlamydial infection among patients with sexually transmitted diseases in India.

BACKGROUND & OBJECTIVES: With increasing burden of human immunodeficiency virus (HIV) infection acquired immunodeficiency syndrome (AIDS) in India, documentation on the epidemiology of genital chlamydial infections in high-risk patients with sexually transmitted diseases (STD) is of significant public health value. Specific diagnosis is essential to prevent the morbidity due to the chlamydial infection and to reduce the risk of acquiring HIV infection. The present study was undertaken to analyse the usefulness of culture and antigen detection by direct fluorescent antibody (DFA) test for assessing the rate of Chlamydia trachomatis infection in symptomatic patients and feasibility of these tests for routine adoption in Indian setting. METHODS: Clinically diagnosed patients of both sex (n=143) attending the Institute of Sexually Transmitted Diseases, Government General Hospital, Chennai who consented for the study, were enrolled. Clinical and demographic details were recorded on a stratified proforma. Genital swab specimens collected from them were subjected for culture using McCoy cell line and for antigen detection by DFA testing. RESULTS: C. trachomatis was isolated in 27 of the total 143 patients (18.9%). Culture positivity was seen in 11 of the 63 (17.5%) males and in 16 of 80 (20%) females. DFA detected C. trachomatis specific antigen in 35 patients (24.5%); 15 (23.8%) males and 20 (25%) females. The rate of C. trachomatis diagnosis increased to 25.2 per cent by adopting both the methods as against 18.9 per cent by culture only and 24.5 per cent by DFA only. No association of C. trachomatis infection with any predictable genitourinary symptom (s), was seen. INTERPRETATION & CONCLUSION: The findings show a high infection rate for C. trachomatis in symptomatic patients with STD. Clinical symptoms alone can be unreliable in specifically predicting infections with C. trachomatis. Specific diagnostic tests need to be recommended for routine inclusion in the STD diagnosis to facilitate risk reduction of HIV infection in STD patients.

Molecular typing of Neisseria gonorrhoeae from hospital and community isolates by restriction fragment length polymorphism.

Strains of Neisseria gonorrhoeae are generally characterized by auxotyping, serotyping, plasmid profile, antibiotic sensitivity and deoxyribonucleic acid (DNA) amplification fingerprinting. The aim of this study was to analyse the generation of restriction fragment length polymorphism (RFLP) patterns by BgIII digestion of total genomic DNA of N. gonorrhoeae isolated from the community (n =30) and the hospital (n =15) and to establish an association with serogrouping and antibiogram. The RFLP patterns produced by BgIII restriction digestion showed 7 different patterns among 30 community isolates and 9 different patterns among 15 hospital isolates. 66.7% of isolates belonged to serogroup WI. Penicillin resistance was observed in 46.7% of community isolates and 66.7% hospital isolates. However, penicillinase producing N. gonorrhoeae (PPNG) were lower in the community (6.6%) than in the hospital isolates (53.3%). PPNG strains were more often seen in serogroup WI. This is the first Indian report on RFLP genotype pattern in N. gonorrhoeae. We noted differences in RFLP genotypes of the community (RFLP types 1, 2, 3, 4, 5 and 7) and hospital strains (RFLP types 6 and 8), while no differences in the serogroup were observed. Ciprofloxacin resistance was 20.0% and 26.6% in the community and hospital isolates, respectively. Ceftriaxone emerges as the current drug of choice for an effective policy of antibiotic treatment of gonorrhoea through syndromic management in developing countries.

Expression pattern of the type 1 sigma receptor in the brain and identity of critical anionic amino acid residues in the ligand-binding domain of the receptor.

The type 1 sigma receptor (sigmaR1) has been shown to participate in a variety of functions in the central nervous system. To identify the specific regions of the brain that are involved in sigmaR1 function, we analyzed the expression pattern of the receptor mRNA in the mouse brain by in situ hybridization. SigmaR1 mRNA was detectable primarily in the cerebral cortex, hippocampus, and Purkinje cells of cerebellum. To identify the critical anionic amino acid residues in the ligand-binding domain of sigmaR1, we employed two different approaches: chemical modification of anionic amino acid residues and site-directed mutagenesis. Chemical modification of anionic amino acids in sigmaR1 with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide reduced the ligand-binding activity markedly. Since it is known that a splice variant of this receptor which lacks exon 3 does not have the ability to bind sigma ligands, the ligand-binding domain with its critical anionic amino acid residues is likely to be present in or around the region coded by exon 3. Therefore, each of the anionic amino acids in this region was mutated individually and the influence of each mutation on ligand binding was assessed. These studies have identified two anionic amino acids, D126 and E172, that are obligatory for ligand binding. Even though the ligand-binding function was abolished by these two mutations, the expression of these mutants was normal at the protein level. These results show that sigmaR1 is expressed at high levels in specific areas of the brain that are involved in memory, emotion and motor functions. The results also provide important information on the chemical nature of the ligand-binding site of sigmaR1 that may be of use in the design of sigmaR1-specific ligands with potential for modulation of sigmaR1-related brain functions.

Na+ - and Cl- -coupled active transport of nitric oxide synthase inhibitors via amino acid transport system B(0,+).

Nitric oxide synthase (NOS) inhibitors have therapeutic potential in the management of numerous conditions in which NO overproduction plays a critical role. Identification of transport systems in the intestine that can mediate the uptake of NOS inhibitors is important to assess the oral bioavailability and therapeutic efficacy of these potential drugs. Here, we have cloned the Na+ - and Cl- -coupled amino acid transport system B(0,+) (ATB(0,+)) from the mouse colon and investigated its ability to transport NOS inhibitors. When expressed in mammalian cells, ATB(0,+) can transport a variety of zwitterionic and cationic amino acids in a Na+ - and Cl- -coupled manner. Each of the NOS inhibitors tested compete with glycine for uptake through this transport system. Furthermore, using a tritiated analog of the NOS inhibitor N(G)-nitro-L-arginine, we showed that Na+ - and Cl- -coupled transport occurs via ATB(0,+). We then studied transport of a wide variety of NOS inhibitors in Xenopus laevis oocytes expressing the cloned ATB(0,+) and found that ATB(0,+) can transport a broad range of zwitterionic or cationic NOS inhibitors. These data represent the first identification of an ion gradient-driven transport system for NOS inhibitors in the intestinal tract.

Na+- and Cl--coupled active transport of carnitine by the amino acid transporter ATB(0,+) from mouse colon expressed in HRPE cells and Xenopus oocytes.

1. ATB(0,+) is an amino acid transporter energized by transmembrane gradients of Na+ and Cl(-) and membrane potential. We cloned this transporter from mouse colon and expressed the clone functionally in mammalian (human retinal pigment epithelial, HRPE) cells and Xenopus laevis oocytes to investigate the interaction of carnitine and its acyl esters with the transporter. 2. When expressed in mammalian cells, the cloned ATB(0,+) was able to transport carnitine, propionylcarnitine and acetylcarnitine. The transport process was Na(+) and Cl(-) dependent and inhibitable by the amino acid substrates of the transporter. The Michaelis constant for carnitine was 0.83 +/- 0.08 mM and the Hill coefficient for Na(+) activation was 1.6 +/- 0.1. 3. When expressed in Xenopus laevis oocytes, the cloned ATB(0,+) was able to induce inward currents in the presence of carnitine and propionylcarnitine under voltage-clamped conditions. There was no detectable current in the presence of acetylcarnitine. Carnitine-induced currents were obligatorily dependent on the presence of Na(+) and Cl(-). The currents were saturable with carnitine and the Michaelis constant was 1.8 +/- 0.4 mM. The analysis of Na(+)- and Cl(-)-activation kinetics revealed that 2 Na(+) and 1 Cl(-) were involved in the transport of carnitine via the transporter. 4. These studies describe the identification of a novel function for the amino acid transporter ATB(0,+). Since this transporter is expressed in the intestinal tract, lung and mammary gland, it is likely to play a significant role in the handling of carnitine in these tissues. 5. A Na(+)-dependent transport system for carnitine has already been described. This transporter, known as OCTN2 (novel organic cation transporter 2), is expressed in most tissues and transports carnitine with high affinity. It is energized, however, only by a Na(+) gradient and membrane potential. In contrast, ATB(0,+) is a low-affinity transporter for carnitine, but exhibits much higher concentrative capacity than OCTN2 because of its energization by transmembrane gradients of Na(+) and Cl(-) as well as by membrane potential.

Structure, function, and tissue expression pattern of human SN2, a subtype of the amino acid transport system N.

We have cloned a new subtype of the amino acid transport system N from a human liver cell line. This transporter, designated SN2, consists of 472 amino acids and exhibits 62% identity with human SN1 at the level of amino acid sequence. SN2-specific transcripts are expressed predominantly in the stomach, brain, liver, lung, and intestinal tract. The sizes of the transcripts vary in different tissues, indicating tissue-specific alternative splicing of the SN2 mRNA. In contrast, SN1 is expressed primarily in the brain and liver and there is no evidence for the presence of multiple transcripts of varying size for SN1. When expressed in mammalian cells, the cloned human SN2 mediates Na(+)-coupled transport of system N-specific amino acid substrates (glutamine, asparagine, and histidine). In addition, SN2 also transports serine, alanine, and glycine. Anionic amino acids, cationic amino acids, imino acids, and N-alkylated amino acids are not recognized as substrates by human SN2. The SN2-mediated transport process is Li(+)-tolerant and highly pH-dependent. The Michaelis-Menten constant for histidine uptake via human SN2 is 0.6 +/- 0.1 mM. The gene coding for SN2 is located on human chromosome Xp11.23. Successful cloning of SN2 provides the first molecular evidence for the existence of subtypes within the amino acid transport system N in mammalian tissues.

Structure and function of ATA3, a new subtype of amino acid transport system A, primarily expressed in the liver and skeletal muscle.

To date, two different transporters that are capable of transporting alpha-(methylamino)isobutyric acid, the specific substrate for amino acid transport system A, have been cloned. These two transporters are known as ATA1 and ATA2. We have cloned a third transporter that is able to transport the system A-specific substrate. This new transporter, cloned from rat skeletal muscle and designated rATA3, consists of 547 amino acids and has a high degree of homology to rat ATA1 (47% identity) and rat ATA2 (57% identity). rATA3 mRNA is present only in the liver and skeletal muscle. When expressed in Xenopus laevis oocytes, rATA3 mediates the transport of alpha-[(14)C](methylamino)isobutyric acid and [(3)H]alanine. With the two-microelectrode voltage clamp technique, we have shown that exposure of rATA3-expressing oocytes to neutral, short-chain aliphatic amino acids induces inward currents. The amino acid-induced current is Na(+)-dependent and pH-dependent. Analysis of the currents with alanine as the substrate has shown that the K(0. 5) for alanine (i.e., concentration of the amino acid yielding half-maximal current) is 4.2+/-0.1 mM and that the Na(+):alanine stoichiometry is 1:1.

cDNA structure, genomic organization, and promoter analysis of the mouse intestinal peptide transporter PEPT1.

We describe in this report the cDNA structure, functional characteristics, genomic organization, and promoter analysis of the mouse H(+)-coupled low-affinity peptide transporter PEPT1. The mouse PEPT1 cDNA cloned from a kidney cDNA library is approximately 3.1 kb long and encodes a protein of 709 amino acids. When expressed heterologously in mammalian cells and in Xenopus laevis oocytes, mouse PEPT1 mediates H(+)-coupled electrogenic transport of the dipeptide glycylsarcosine. The mouse pept1 gene, cloned from a genomic DNA library in bacterial artificial chromosome, is approximately 38 kb long and consists of 23 exons and 22 introns. 5'-Rapid amplification of cDNA ends with poly(A)(+) RNA from mouse intestine has identified the transcription start site that lies 31 bp upstream of the translation start site. The promoter region upstream of the transcription start site does not contain the TATA box but possesses three GC boxes which are the binding sites for the transcription activator SP1. Functional analysis of the promoter region using the luciferase reporter assay in Caco-2 cells (a human intestinal cell line that express PEPT1 constitutively) and five different 5'-deletion fragments of the promoter has shown that essential promoter/enhancer elements are present within 1140 bp upstream of the transcription start site.

Structure, function, and regional distribution of the organic cation transporter OCT3 in the kidney.

We examined in this study the expression of the potential-sensitive organic cation transporter OCT3 in the kidney. A functionally active OCT3 was cloned from a mouse kidney cDNA library. The cloned transporter was found to be capable of mediating potential-dependent transport of a variety of organic cations including tetraethylammonium. This function was confirmed in two different heterologous expression systems involving mammalian cells and Xenopus laevis oocytes. We have also isolated the mouse OCT3 gene and deduced its structure and organization. The OCT3 gene consists of 11 exons and 10 introns. In situ hybridization studies in the mouse kidney have shown that OCT3 mRNA is expressed primarily in the cortex. The expression is evident in the proximal and distal convoluted tubules. The expression of OCT3 in human kidney was confirmed by RT-PCR. We have also cloned OCT3 from human placenta and human kidney. Human OCT3 exhibits 86% identity with mouse OCT3 in amino acid sequence. Human OCT3 was found to transport tetraethylammonium and a variety of other organic cations. The transport process was electrogenic. We conclude that OCT3 is expressed in mammalian kidney and that it plays an important role in the renal clearance of cationic drugs.

Transport of valganciclovir, a ganciclovir prodrug, via peptide transporters PEPT1 and PEPT2.

In clinical trials, valganciclovir, the valyl ester of ganciclovir, has been shown to enhance the bioavailability of ganciclovir when taken orally by patients with cytomegalovirus infection. We investigated the role of the intestinal peptide transporter PEPT1 in this process by comparing the interaction of ganciclovir and valganciclovir with the transporter in different experimental systems. We also studied the interaction of these two compounds with the renal peptide transporter PEPT2. In cell culture model systems using Caco-2 cells for PEPT1 and SKPT cells for PEPT2, valganciclovir inhibited glycylsarcosine transport mediated by PEPT1 and PEPT2 with K(i) values (inhibition constant) of 1.68+/-0.30 and 0.043+/- 0.005 mM, respectively. The inhibition by valganciclovir was competitive in both cases. Ganciclovir did not interact with either transporter. Similar studies done with cloned PEPT1 and PEPT2 in heterologous expression systems yielded comparable results. The transport of valganciclovir via PEPT1 was investigated directly in PEPT1-expressing Xenopus laevis oocytes with an electrophysiological approach. Valganciclovir, but not ganciclovir, induced inward currents in PEPT1-expressing oocytes. These results demonstrate that the increased bioavailability of valganciclovir is related to its recognition as a substrate by the intestinal peptide transporter PEPT1. This prodrug is also recognized by the renal peptide transporter PEPT2 with high affinity.

Cloning of an amino acid transporter with functional characteristics and tissue expression pattern identical to that of system A.

We report here on the cloning and functional characterization of the protein responsible for the system A amino acid transport activity that is known to be expressed in most mammalian tissues. This transporter, designated ATA2 for amino acid transporter A2, was cloned from rat skeletal muscle. It is distinct from the neuron-specific glutamine transporter (GlnT/ATA1). Rat ATA2 consists of 504 amino acids and bears significant homology to GlnT/ATA1 and system N (SN1). ATA2-specific mRNA is ubiquitously expressed in rat tissues. When expressed in mammalian cells, ATA2 mediates Na(+)-dependent transport of alpha-(methylamino)isobutyric acid, a specific model substrate for system A. The transporter is specific for neutral amino acids. It is pH-sensitive and Li(+)-intolerant. The Na(+):amino acid stoichiometry is 1:1. When expressed in Xenopus laevis oocytes, transport of neutral amino acids via ATA2 is associated with inward currents. The substrate-induced current is Na(+)-dependent and pH-sensitive. The amino acid transport system A is particularly known for its adaptive and hormonal regulation, and therefore the successful cloning of the protein responsible for this transport activity represents a significant step toward understanding the function and expression of this transporter in various physiological and pathological states.

beta-lactam antibiotics as substrates for OCTN2, an organic cation/carnitine transporter.

Therapeutic use of cephaloridine, a beta-lactam antibiotic, in humans is associated with carnitine deficiency. A potential mechanism for the development of carnitine deficiency is competition between cephaloridine and carnitine for the renal reabsorptive process. OCTN2 is an organic cation/carnitine transporter that is responsible for Na(+)-coupled transport of carnitine in the kidney and other tissues. We investigated the interaction of several beta-lactam antibiotics with OCTN2 using human cell lines that express the transporter constitutively as well as using cloned human and rat OCTN2s expressed heterologously in human cell lines. The beta-lactam antibiotics cephaloridine, cefoselis, cefepime, and cefluprenam were found to inhibit OCTN2-mediated carnitine transport. These antibiotics possess a quaternary nitrogen as does carnitine. Several other beta-lactam antibiotics that do not possess this structural feature did not interact with OCTN2. The interaction of cephaloridine with OCTN2 is competitive with respect to carnitine. Interestingly, many of the beta-lactam antibiotics that were not recognized by OCTN2 were good substrates for the H(+)-coupled peptide transporters PEPT1 and PEPT2. In contrast, cephaloridine, cefoselis, cefepime, and cefluprenam, which were recognized by OCTN2, did not interact with PEPT1 and PEPT2. The interaction of cephaloridine with OCTN2 was Na(+)-dependent, whereas the interaction of cefoselis and cefepime with OCTN2 was largely Na(+)-independent. Furthermore, the Na(+)-dependent, OCTN2-mediated cellular uptake of cephaloridine could be demonstrated by direct uptake measurements. These studies show that OCTN2 plays a crucial role in the pharmacokinetics and therapeutic efficacy of certain beta-lactam antibiotics such as cephaloridine and that cephaloridine-induced carnitine deficiency is likely to be due to inhibition of carnitine reabsorption in the kidney.

Carnitine transport and its inhibition by sulfonylureas in human kidney proximal tubular epithelial cells.

The kidney plays an important role in the homeostasis of carnitine by its ability to reabsorb carnitine almost completely from the glomerular filtrate. The transport process responsible for this reabsorption has been investigated thus far only in laboratory animals. Here we report on the characteristics of carnitine uptake in a proximal tubular epithelial cell line derived from human kidney. The uptake process was found to be obligatorily dependent on Na+ with no involvement of anions. The process was saturable, with a Michaelis-Menten constant of 14 +/- 1 microM. The Na+:carnitine stoichiometry was 1:1. The same process also was found to be responsible for the uptake of acetylcarnitine and propionylcarnitine, two acyl esters of carnitine with potential for therapeutic use in humans. The uptake process was specific for carnitine and its acyl esters. Betaine, a structural analog of carnitine, interacted with the uptake process to a significant extent. The present studies also showed that sulfonylureas, oral hypoglycemic agents currently used in the management of type 2 diabetes, inhibited the carnitine uptake system. Among the sulfonylureas tested, glibenclamide was the most potent inhibitor. The inhibition was competitive. Glibenclamide inhibited the uptake not only of carnitine but also of acetylcarnitine and propionylcarnitine. The inhibition most likely was the result of direct interaction of the compound with the carnitine transporter because the inhibition could be demonstrated in purified rat kidney brush border membrane vesicles.