Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter.

Primary systemic carnitine deficiency (SCD; OMIM 212140) is an autosomal recessive disorder characterized by progressive cardiomyopathy, skeletal myopathy, hypoglycaemia and hyperammonaemia. SCD has also been linked to sudden infant death syndrome. Membrane-physiological studies have suggested a defect of the carnitine transport system in the plasma membrane in SCD patients and in the mouse model, juvenile visceral steatosis. Although the responsible loci have been mapped in both human and mouse, the underlying gene has not yet been identified. Recently, we cloned and analysed the function of a novel transporter protein termed OCTN2. Our observation that OCTN2 has the ability to transport carnitine in a sodium-dependent manner prompted us to search for mutations in the gene encoding OCTN2, SLC22A5. Initially, we analysed the mouse gene and found a missense mutation in Slc22a5 in jvs mice. Biochemical analysis revealed that this mutation abrogates carnitine transport. Subsequent analysis of the human gene identified four mutations in three SCD pedigrees. Affected individuals in one family were homozygous for the deletion of a 113-bp region containing the start codon. In the second pedigree, the affected individual was shown to be a compound heterozygote for two mutations that cause a frameshift and a premature stop codon, respectively. In an affected individual belonging to a third family, we found a homozygous splice-site mutation also resulting in a premature stop codon. These mutations provide the first evidence that loss of OCTN2 function causes SCD.

First Isolation of Bacillus licheniformis from Bovine Mastitis in Iran.

Bacillus licheniformis is a gram-positive, endospore-forming, saprophytic and facultative anaerobe that is resistant to heat and environmental conditions. This study was the first to isolate and confirm B. licheniformis as a cause of bovine mastitis in Iran. In the summer of 2020, 105 samples of mastitic milk were collected from dairy farms around Tehran and sent to the microbiology laboratory of the Faculty of Veterinary Medicine at the University of Tehran. The bacterial pathogens were identified using selective and differential culture media and confirmed by PCR to contain the toxin synthetase genes licA, licB and licC in mastitic isolates of B. licheniformis. Resistance patterns to 19 antibiotics were determined for two isolates of B. licheniformis. Staphylococcus aureus and Escherichia coli were identified as the most important organisms in the samples. B. licheniformis was isolated from the two samples containing all three genes. Both isolates were resistant to streptomycin, trimethoprim-sulfamethoxazole, cefixime, ampicillin, bacitracin, clindamycin, and gentamicin. B. licheniformis was reported for the first time in Iran as a cause of bovine mastitis with clinical symptoms. The first isolation of toxin-producing strains of B. licheniformis from mastitic cows in Iran raises concerns about the safety of dairy products. In principle, selected strains with toxigenic potential should not be used as feed additives and animal feed. However, whole genome sequencing is proposed to search for genes coding for toxins.

Clonal relatedness and antimicrobial susceptibility of Salmonella serovars isolated from humans and domestic animals in Iran: a one health perspective.

Background: Salmonellosis is one of the most important zoonotic diseases in humans and animals worldwide. Aims: The main objective of this study was to report serovars, clonal relatedness, and antimicrobial resistance of Salmonella strains isolated from human, different animal hosts including pigeons, broilers, cattle, camel, parrots, and hamsters in different regions of Iran. Methods: Twenty-four Salmonella isolates were confirmed at the genus level by biochemical tests and polymerase chain reaction (PCR) by showing the presence of invA gene. Serovars were determined and their clonal relatedness was assessed by RAPD-PCR and antibiotic resistance profiles. Results: Overall, Salmonella Typhimurium was the most prevalent serovar (45.8%, 11/24), which was recovered from humans, pigeons, and camels. Salmonella Enteritidis (29.2%, 7/24) was the second common serovar that was recovered from cattle, broilers, humans, and hamsters. Salmonella Infantis (12.5%, 3/24) belonged only to broiler sources, and Salmonella Seftenberg (12.5%, 3/24) was isolated from eggs and a parrot. The major RAPD pattern was VI (33.3%) in which the two S. Typhimurium isolates (belonged to humans and pigeons) exhibited similarity in both RAPD pattern and resistance profile. Antimicrobial susceptibility test showed full resistance to tylosin and erythromycin (100%, 24/24). All isolates (100%, 24/24) were susceptible to ceftriaxone, cefixime, and gentamicin. In total, 75% of the isolates were multi-drug resistant (MDR) and revealed 15 different antimicrobial resistance profiles (R-type). Conclusion: This study supports the potential transmission of Salmonella serovars via animal contacts. Thus, it is necessary to establish a national systematic monitoring program with one health approach for controlling Salmonella infections.

[The inhibitors of the apical sodium-dependent bile acid transporter (ASBT) as promising drugs]. / Ingibitory natrii-zavisimogo perenoschika zhelchnykh kislot (ASBT) kak perspektivnye lekarstvennye sredstva.

Inhibition of the apical sodium-dependent bile acid transporter (ASBT, also known as IBAT - ileal bile acid transporter, SLC10A2) leads to disruption of the enterohepatic circulation of bile acids and their excretion with fecal masses. This is accompanied by cholesterol utilization for synthesis of new bile acids. ASBT inhibitors are promising drugs for the treatment of such diseases as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, type 2 diabetes mellitus, necrotic enterocolitis, chronic constipation, atherosclerosis. To date the most known chemically synthesized inhibitors are: A3309, SHP626, A4250, 264W94, GSK2330672, SC-435. All of them are at different stages of clinical trials, which confirm the high efficacy and good tolerance of these inhibitors. Current trends in this field also include directed chemical synthesis of ASBT inhibitors, as well as their search among substances of plant origin.

Study of an unusual paratyphoid epornitic in canaries (Serinus canaria).

High mortalities in 17 canary flocks from different regions of Tehran, Iran, were reported. Necropsy and histopathologic examination revealed necrotic hepatitis and overall congestive septicaemia in carcasses. Salmonella enterica was isolated from 34 examined samples, two samples from each flock, including visceral organs of carcasses and droppings of live diseased birds. All isolates were typed as Salmonella enterica serovar Typhimurium by conventional serotyping. Antibiotic resistance profiling using 33 antibiotics and random amplification of polymorphic DNA differentiation by three primers were performed and showed an identical clonal relationship between these isolates and S. Typhimurium isolated from a sample of feedstuffs. Changing the feed ingredients along with antibiotic therapy via the drinking water by enrofloxacin solution controlled the outbreaks, and mortalities ceased. The zoonotic nature of S. Typhimurium and close contact of bird owners with pet birds in the home environment made the case significant in relation to public health.

Distribution of Antibiotic Resistance Genes among the Phylogroups of Escherichia coli in Diarrheic Calves and Chickens Affected by Colibacillosis in Tehran, Iran.

Antibiotic resistance occurs in the endogenous flora of exposed population in addition to pathogenic bacteria. This study was conducted to evaluate the distribution of antibiotic resistance genes among 63 isolates of Escherichia coli of Escherichia coli (E. coli) in diarrheic calves and poultry. According to the results, B1 and B2 were the most prevalent phylogroups of E. coli in calves and poultry carcasses, respectively. Antimicrobial resistance was observed in 76% of the isolates, and 62% of the strains were multi-drug resistant. Antibiotic resistance in E. coli strains obtained from calves strains was significantly higher than those obtained from poultries. Additionally, the strains of B1 and D phylogroups had the highest and lowest antimicrobial resistance, respectively. At least one encoding gene for integrone was detected in 23 strains (36.5%) and Class I integron had the highest prevalence. Accordingly, this study gave baseline information on the magnitude of the resistance problem and its genetic background in E. coli from domesticated animals of the Tehran, Iran. Moreover, the power of oligonucleotide array technology in the discrimination of different genotypes during a short time was confirmed in this study.

Carrier-mediated transport of oligopeptides in the human fibrosarcoma cell line HT1080.

To explore the feasibility of targeting human tumor cells via their transport systems, dipeptide uptake was studied in the human fibrosarcoma cell line HT1080 and the human fibroblast cell line IMR-90 by the use of hydrolysis-resistant glycylsarcosine (Gly-Sar). The uptake of [14C]Gly-Sar into HT1080 was time dependent. Kinetic analysis of the concentration dependence of the initial rate of [14C]Gly-Sar uptake showed that a carrier-mediated transport system with a K(m) of 11.4 +/- 3.3 mM and V(max) of 26.8 +/- 4.0 (nmol/15 min/mg protein) and a nonsaturable component (k(d) of 0.80 microl/15 min/mg protein) were responsible for the dipeptide uptake by HT1080 cells. The optimal pH for the maximal uptake was around 6.0. [14C]Gly-Sar uptake was inhibited by various di- and tripeptides and peptide-mimetic drugs, such as bestatin and cefadroxil. [14C]Gly-Sar uptake was not affected by the presence of amino acids or tetra- or pentapeptides. The uptake of cefadroxil was reduced significantly by unlabeled Gly-Sar. Moreover, Gly-Gly and Gly-Leu produced an increase in the apparent K(m) of the uptake of Gly-Sar without altering V(max). On the other hand, dipeptide uptake by IMR-90, which is a normal diploid cell line (not malignant), showed no saturable transport. These results suggest that HT1080 cells take up dipeptides via a pH-dependent transporter. This is the first report showing that a dipeptide transport system, which is similar but not identical to the well-characterized oligopeptide transporters PepT1 and PepT2, exists in fibroblast-derived tumor cells but not in normal fibroblasts. The present finding could be the basis of a novel strategy for the specific delivery of oligopeptide-mimetic anticancer drugs into tumor cells.

Molecular characterization of Salmonella enterica serotype Enteritidis isolates from food and human samples by serotyping, antimicrobial resistance, plasmid profiling, (GTG)5-PCR and ERIC-PCR.

In recent years, Salmonella enterica serovar Enteritidis has been a primary cause of human salmonellosis in many countries. The major objective of this study was to investigate genetic diversity among Salmonella Enteritidis strains from different origins (food and human) by Enterobacterial Repetitive Intergenic Consensus (ERIC) -PCR, as well as to assess their plasmid profiling and antimicrobial resistance. A total of 30 Salmonella Enteritidis isolates, 15 from food samples (chicken, lamb, beef and duck meats) and 15 from clinical samples were collected in Tehran. Identification of isolates as Salmonella was confirmed by using conventional standard biochemical and serological tests. Multiplex-PCR was used for serotyping of isolates to identify Salmonella Enteritidis. Antimicrobial susceptibility testing to 16 agents founds drug resistance patterns among Salmonella Enteritidis isolates. No resistance was observed to cephalexin, ceftriaxone, ceftazidime and cefotaxime, ciprofloxacin, imipenem or meropenem, chloramphenicol and gentamicin. The highest resistance (96.7%) was observed to nitrofurantoin. Seven plasmid profiles (P1-P7) were detected, and a 68-kb plasmid was found in all isolates. Two different primers; ERIC and (GTG)5 were used for genotyping, which each produced four profiles. The majority of clinical and food isolates fell into two separate common types (CTs) with a similar percentage of 95% by ERIC-PCR. Using primer (GTG)5, 29 isolates incorporated in three CTs with 70% of isolates showing a single banding pattern. Limited genetic diversity among human and food isolates of Salmonella Enteritidis may indicate that contaminated foods were possibly the source of human salmonellosis. These results confirmed that ERIC-PCR genotyping has limited discriminatory power for Salmonella Enteritidis of different origin.

Anion antiport mechanism is involved in transport of lactic acid across intestinal epithelial brush-border membrane.

Intestinal epithelial membrane transport of L-lactic acid was characterized using rabbit jejunal brush-border membrane vesicles (BBMVs). The uptake of L-[(14)C]lactic acid by BBMVs showed an overshoot phenomenon in the presence of outward-directed bicarbonate and/or inward-directed proton gradients. Kinetic analysis of L-[(14)C]lactic acid uptake revealed the involvement of two saturable processes in the presence of both proton and bicarbonate gradients. An arginyl residue-modifying agent, phenylglyoxal, inhibited L-[(14)C]lactic acid transport by the proton cotransporter, but not by the anion antiporter. The initial uptakes of L-[(14)C]lactic acid which are driven by bicarbonate ion and proton gradients were inhibited commonly by monocarboxylic acids and selectively by anion exchange inhibitor 4, 4'-diisothiocyanostilbene-2,2'-disulfonic acid and protonophore carbonylcyanide p-trifluoromethoxyphenylhydrazone, respectively. These observations demonstrate that L-lactic acid is transported across the intestinal brush-border membrane by multiple mechanisms, including an anion antiporter and a previously known proton cotransporter.

Na(+)-coupled transport of L-carnitine via high-affinity carnitine transporter OCTN2 and its subcellular localization in kidney.

The mechanism of Na(+)-dependent transport of L-carnitine via the carnitine/organic cation transporter OCTN2 and the subcellular localization of OCTN2 in kidney were studied. Using plasma membrane vesicles prepared from HEK293 cells that were stably transfected with human OCTN2, transport of L-carnitine via human OCTN2 was characterized. Uptake of L-[(3)H]carnitine by the OCTN2-expressing membrane vesicles was significantly increased in the presence of an inwardly directed Na(+) gradient, with an overshoot, while such transient uphill transport was not observed in membrane vesicles from cells that were mock transfected with expression vector pcDNA3 alone. The uptake of L-[(3)H]carnitine was specifically dependent on Na(+) and the osmolarity effect showed that Na(+) significantly influenced the transport rather than the binding. Changes of inorganic anions in the extravesicular medium and of membrane potential by valinomycin altered the initial uptake activity of L-carnitine by OCTN2. In addition, the fluxes of L-carnitine and Na(+) were coupled with 1:1 stoichiometry. Accordingly, it was clarified that Na(+) is coupled with flux of L-carnitine and the flux is an electrogenic process. Furthermore, OCTN2 was localized on the apical membrane of renal tubular epithelial cells. These results clarified that OCTN2 is important for the concentrative reabsorption of L-carnitine after glomerular filtration in the kidney.

Sequence, tissue distribution and developmental changes in rat intestinal oligopeptide transporter.

Complementary DNA clones encoding the rat PepT1 small-intestinal oligopeptide transporter were isolated from a jejunal library by cross-hybridization with a rabbit PepT1 cDNA probe. The cDNA sequence indicates that rat PepT1 is composed of 710 amino acids and shows 77% and 83% amino acid sequence identity with rabbit and human PepT1, respectively. Northern blot analysis detected rat PepT1 mRNA in the small intestine and kidney. Intestinal PepT1 mRNA levels were highest in 4-day old rats, and then decreased reaching the adult level by day 28 after birth. These results indicate that the expressions of PepT1 gene change markedly during development.

The effect of antisera to insulin, 2-deoxyglucose-induced hyperglycemia, and starvation on wound healing in normal mice.

Wound healing was examined in normal C57BL/6 male mice treated with antiserum to insulin or 2-deoxyglucose (2-DG) and in mice starved for 33 h. Hyperglycemia was induced after antiserum or 2-DG treatment; the blood glucose was lowered in the starved mice when compared with controls. Small dermal wounds were made in the ears of the mice 1 h after the initial injection of antisera or 2-DG. The starved group were wounded after 25 h of fasting. All animals were biopsied 8 h later. The wounds were examined by light microscopy and wound components (capillaries, fibroblasts, PMNs, collagen, and edema) were quantitated by lineal point analysis. Mice treated with antisera to insulin and mice starved for 33 h had an impaired healing response; the mice treated with 2-DG had a response similar to controls. These results suggest that hyperglycemia, per se, or the production of any toxic metabolites from high blood glucose levels could not alone induce the poor healing response. The depressed response in the antiserum-treated and starved mice may be due to the decreased availability of insulin to the wound tissues. These data support the hypotheses that insulin is a necessary component for an adequate wound healing response. In addition to a role in glucose transport and metabolism, insulin may also promote cellular growth.

Induction of hyperglycemia with insulin antibodies to B-chain determinants.

Insulin antibodies measured by a radioimmune method (ABR) are significantly better inducers of hyperglycemia than are insulin antibodies measured by an immune hemolysis method (ABH) when injected intraperitoneally into mice. The ability to induce hyperglycemia by an insulin antiserum can be predicted by the titer of ABR measured. ABR interact in vitro with determinants severely perturbed on nickel-insulin, partially perturbed on proinsulin and desasparagine-desalanine insulin, and unaffected on zinc-insulin or zinc-free monocomponent insulin. ABH, on the other hand, interact in vitro with determinants severely perturbed on proinsulin and desasparagine-desalanine insulin but stabilized on nickel-insulin and zinc-insulin. Since the connecting peptide of proinsulin is probably in apposition to the A-chain residues on the solvent surface, the more effective reaction of proinsulin with ABR than with ABH is submitted as evidence that ABR are directed toward residues on the B-chain surface of insulin. Because ABR are more effective inducers of hyperglycemia than are ABH, it is proposed that the degree of hyperglycemia induced by antibodies in vivo is a result of interactions with determinants on the B-chain surface of insulin. These results support the possibility that insulin in vivo is more accessible for interaction with antibodies directed to the B-chain of insulin. It is also possible that ABR, which are directed to B-chain determinants, are of higher affinity than is the affinity between insulin and receptors or that the active site of insulin for maintaining euglycemia includes the B-chain surface residues.

Variability of serum phenytoin concentrations in nursing home patients.

A marked variability in serum phenytoin concentrations was observed in an elderly nursing home resident. To determine the prevalence of this problem, 15 frail nursing home patients who were receiving phenytoin therapy were studied over a mean follow-up period of 10.6 +/- 0.89 months. The mean number of serum phenytoin level measurements during this period was 13.1 +/- 1.5. For a given individual, there was no variation in phenytoin dose or preparation administered. All the patients had a difference of more than 50% between the highest and the lowest serum phenytoin levels, and in five patients (33%), the difference exceeded 150%. The change in serum phenytoin level was temporally related to influenza vaccination in only three patients. The form of phenytoin was not a significant determinant of the variability in this patient population, nor did enteral feeding have any effect. It is recommended that nursing home patients receiving phenytoin therapy have periodic serum phenytoin measurements obtained, even in the absence of seizures or classic signs of phenytoin toxicity.

High-dose short-term administration of naringin did not alter talinolol pharmacokinetics in humans.

Naringin is considered the major causative ingredient of the inhibition of intestinal drug uptake by grapefruit juice. Moreover, it is contained in highly dosed nutraceuticals available on the market. A controlled, open, randomized, crossover study was performed in 10 healthy volunteers to investigate the effect of high-dose naringin on the bioavailability of talinolol, a substrate of intestinal organic anion-transporting polypeptide (OATP)-mediated uptake. Following 6-day supplementation with 3 capsules of 350 mg naringin daily, 100mg talinolol were administered orally with 3 capsules of the same dietary supplement (1050 mg naringin) on the seventh day. This test treatment was compared to 100mg talinolol only (control). The results showed that short-term high-dose naringin supplementation did not significantly affect talinolol pharmacokinetics. Geometric mean ratios of test versus control ranged between 0.90 and 0.98 for talinolol c(max), AUC(0-48 h), AUC(0-∞), t(1/2) and A(e(0-48 h)). The high dose may provoke inhibition of the efflux transporter P-glycoprotein (P-gp) which counteracts the uptake inhibition. As disintegration and dissolution processes are required for the solid dosage form, dissolved naringin may arrive at the site of interaction after talinolol is already absorbed. In conclusion, the effect of nutraceuticals on drug pharmacokinetics can deviate from that observed when administered as food component due to the different dose and dosage form.

Molecular identification of Salmonella Infantis isolated from backyard chickens and detection of their resistance genesby PCR.

This study aims at molecular identification of Salmonella Infantis isolated from backyard chickens and the detection of their antibiotic resistance genes. A total of 46 Salmonella-suspected samples isolated from backyard chickens of northern Iran were collected. Serotyping was done by the traditional method and then confirmed by PCR. Antimicrobial susceptibility of the isolates against 13 antimicrobial agents was determined by the standard disk diffusion method. There were 44 samples identified as Salmonella. Serotyping results showed that all 44 isolates belonged to serogroup C1 and serovar Infantis. The most resistance observed was to tetracycline and doxycycline (100%), chloramphenicol (79%) and florfenicol (72%). The floR, catI, tetA and tetG genes were used for the detection of florfenicol chloramphenicol and tetracycline resistance. In order to identify the phenotypic resistance in strains which showed resistance genes by PCR, colony PCR and culture on plates each containing antibiotic was performed simultaneously. All the Salmonella Infantis resistant to florfenicol and chloramphenicol harbored floR and catI. None of the Salmonella resistant to tetracycline carried tetA or tetG. The result of colony PCR and culture in antibiotic medium confirmed the results of PCR and indicated phenotypic resistance in these samples.

Two novel missense mutations of the OCTN2 gene (W283R and V446F) in a patient with primary systemic carnitine deficiency.

Primary systemic carnitine deficiency (SCD) is an autosomal recessive disorder of fatty acid oxidation caused by defective cellular carnitine transport. The disease is characterized by metabolic derangement simulating Reye's syndrome, hypoglcaemia, progressive cardiomyopathy and skeletal myopathy. Recently, it was shown that SCD is caused by mutations in the organic cation/carnitine transporter OCTN2 (SLC22A5). We report two novel mutations, W283R and V446F, which are both missense mutations in an affected infant. In vitro expression studies demonstrated that both are actually function-loss mutations with virtually no uptake activity. This is the first report of compound heterozygosity for two missense mutations in a patient with SCD. Hum Mutat 15:118, 2000.

Brain-to-blood active transport of beta-alanine across the blood-brain barrier.

A high-affinity antiluminal uptake system for beta-alanine was demonstrated in primary cultured bovine brain capillary endothelial cells (BCEC) for which K(t) is 66.9 microM. beta alanine uptake was energy-, sodium- and chloride ion-dependent. beta-amino acids strongly inhibited the uptake, while alpha- and gamma-amino acids had a little or no inhibitory effect. In ATP-depleted cells, the uptake was stimulated by preloading beta-alanine or taurine but not by L-leucine. These results suggest that beta-alanine is actively transported across the antiluminal membrane of BCECs that is common to beta-amino acids. The system may function for the efflux from the brain to blood.

Cloning and characterization of a novel human pH-dependent organic cation transporter, OCTN1.

cDNA for a novel proton/organic cation transporter, OCTN1, was cloned from human fetal liver and its transport activity was investigated. OCTN1 encodes a 551-amino acid protein with 11 transmembrane domains and one nucleotide binding site motif. It is strongly expressed in kidney, trachea, bone marrow and fetal liver and in several human cancer cell lines, but not in adult liver. When expressed in HEK293 cells, OCTN1 exhibited saturable and pH-dependent [3H]tetraethyl ammonium uptake with higher activity at neutral and alkaline pH than at acidic pH. Furthermore, treatment with metabolic inhibitors reduced the uptake, which is consistent with the presence of the nucleotide binding site sequence motif. Although its subcellular localization and detailed functional characteristics are not clear at present, OCTN1 appears to be a novel proton antiporter that functions for active secretion of cationic compounds across the renal epithelial brush-border membrane. It may play a role in the renal excretion of xenobiotics and their metabolites.

Immunolocalization and pharmacological relevance of oligopeptide transporter PepT1 in intestinal absorption of beta-lactam antibiotics.

A polyclonal antibody (anti-PepT1/C) was raised against the rabbit intestinal H(+)-coupled oligopeptide transporter, PepT1. Anti-PepT1/C detected 70-80-kDa protein in crude membranes obtained from rabbit duodenum, jejunum and ileum. PepT1 was localized in the brush-border of the absorptive epithelial cells by subcellular fractionation of membranes on a sucrose density gradient and by immunohistochemistry using light and electron microscopy. Transport activity for cephalosporins and dipeptide expressed in Xenopus laevis oocytes injected with total mRNA obtained from rabbit small intestine was eliminated completely by prehybridization of the mRNA with antisense oligonucleotide against the 5'-coding region of rabbit PepT1 cDNA.