Evaluation of the GenoType® MTBDRsl assay for susceptibility testing of second-line anti-tuberculosis drugs.

BACKGROUND: The GenoType® MTBDRsl assay is a new rapid assay for the detection of resistance to second-line anti-tuberculosis drugs. OBJECTIVE: To evaluate the MTBDRsl assay on 342 multidrug-resistant tuberculosis isolates for resistance to ofloxacin (OFX), kanamycin (KM), capreomycin (CPM) and ethambutol (EMB), to compare the results to the agar proportion method, and to test discrepant results using DNA sequencing. RESULT: The sensitivity and specificity of the MTBDRsl assay were respectively 70.3% and 97.7% for OFX, 25.0% and 98.7% for KM, 21.2% and 98.7% for CPM and 56.3% and 56.0% for EMB. DNA sequencing identified mutations that were not detected by the MTBDRsl assay. The 8/11 phenotypically OFX-resistant isolates had mutations in gyrA (2/8 had an additional mutation in the gyrB gene), 1/11 had mutations only in the gyrB gene, 6/21 phenotypically KM-resistant isolates had mutations in the rrs gene, and 7/26 and 20/26 phenotypically CPM-resistant isolates had mutations in the rrs and tlyA genes. CONCLUSION: The MTBDRsl assay showed lower sensitivity than previous studies. The assay performed favourably for OFX; however, it was less sensitive in the detection of KM/CPM resistance and demonstrated low sensitivity and specificity for EMB resistance. It is recommended that the MTBDRsl assay include additional genes to achieve better sensitivity for all the drugs tested.

Molecular mechanism of the intestinal biotin transport process.

Previous studies have characterized different aspects of the cellular/membrane mechanism and regulation of the intestinal uptake process of the water-soluble vitamin biotin. Little, however, is known about the molecular mechanisms of the uptake process. In this study, we have identified a cDNA from rat small intestine that appears to be involved in biotin transport. The open reading frame of this cloned cDNA consisted of 1,905 bases and was identical to that identified for the vitamin transporter in placental tissue. Significant heterogeneity, however, was found in the 5' untranslated region of this clone, with three distinct variants (II, III, IV) being identified in the small intestine; the placental variant (variant I), however, was not present in the small gut. Variant II was found to be the predominant form expressed in the rat small and large intestines. Functional identity of the cloned intestinal cDNA was confirmed by stable expression in COS-7 cells, which showed a four- to fivefold increase in biotin uptake in transfected COS-7 cells compared with controls. The induced biotin uptake in transfected COS-7 cells was found to be 1) Na(+) dependent, 2) saturable as a function of concentration with an apparent K(m) of 8. 77 microM and a V(max) of 779.7 pmol. mg protein(-1). 3 min(-1), and 3) inhibited by unlabeled biotin and pantothenic acid and their structural analogs. The distribution of complementary mRNA transcripts of the cloned cDNA along the vertical and longitudinal axes of the intestinal tract was also determined. Results of this study describe the molecular characteristics of the intestinal biotin absorption process and report the identification of a cDNA that encodes a Na(+)-dependent biotin uptake carrier that appears to exist in the form of multiple variants.

Human intestinal folate transport: cloning, expression, and distribution of complementary RNA.

BACKGROUND & AIMS: Despite intensive investigations, very little is known about the molecular identity(ies) of the intestinal folate transport system(s), especially in humans. The aim of this study was to isolate a functional human intestinal folate carrier complementary DNA (cDNA) clone and determine the distribution of complementary RNA at the tissue and cellular levels. METHODS: Hybridization screening, modified Marathon cDNA amplification, expression in Xenopus oocytes, Northern analysis, and in situ hybridization were used. RESULTS: The hIFC-1 cDNA contains an open reading frame for 591 amino acids (relative molecular mass = 64,826, pI = 9.4, 12 transmembrane domains, three protein kinase C phosphorylation sites, and one N-glycosylation site) with 74% DNA and 66% amino acid sequence homologies with the mouse cDNA counterpart. Xenopus oocytes injected with hIFC-1 cRNA show induced folate uptake that was (1) saturable with substrate concentration (apparent Michaelis constant = 0.71 +/- 0.06 micromol/L; maximum velocity = 128 +/- 3 fmol x h(-1) x oocyte(-1)), (2) inhibited by methotrexate, folinic acid, and folic acid (Ki = 0.84 micromol/L, 0.71 micromol/L, and 10 micromol/L, respectively), and (3) sensitive to 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (Ki = 0.29 mmol/L). Northern analysis showed wide distribution of hIFC1-complementary messenger RNA species in various human tissues. In situ hybridization on sections of human jejunum showed preferential hIFC-1 expression in epithelial cells, especially in the upper half of the villi. CONCLUSIONS: These results represent the first molecular characterization of a human small intestinal folate carrier.

Intestinal folate transport: identification of a cDNA involved in folate transport and the functional expression and distribution of its mRNA.

Although the mechanism of folate intestinal transport has been the subject of intensive studies, very little is known about the molecular identity of the transport system(s) involved. In this investigation, we screened a mouse intestinal cDNA library using as probe the cDNA clone of a reduced folate carrier (RFC1) of mouse leukemia L1210 cells, and identified a positive clone, IFC1(RFC1). The cloned cDNA consisted of 2274 base pairs with an open reading frame that encodes a putative polypeptide of 512 amino acids with a predicted molecular mass of 58,112 daltons and 12 putative transmembrane domains. The polypeptide appears to carry a net positive charge (pI = 8.6) which may be important for its interaction with the negatively charged substrate. Functional identity of the IFC1(RFC1) clone was established by expression in Xenopus oocytes. An 11-fold increase in 5-methyltetrahydrofolate (5-MTHF) uptake was observed in oocytes injected with 10 ng IFC1(RFC1) cRNA compared to water-injected controls. The expressed folate uptake in the cRNA injected oocyte was (1) 4,4'-diisothiocyanatosilbene-2,2'-disulfonic acid (DIDS)-sensitive; and (2) saturable with an apparent Km of 1.99 +/- 0.32 micrometers and a V(max) of 3782 +/- 188 fmol/oocyte per h. The distribution of mRNA species complementary to IFC1(RFC1) in different mouse tissues was examined by Northern blot analysis. In addition to the small intestine, expression of such mRNA species were also found in the kidney, large intestine, brain, heart and liver. Furthermore, mRNA species complementary to IFC1(RFC1) were also detected by Northern blot analysis in the small intestine of human and other animal species (rat and rabbit). Expression of mRNA complementary to IFC1(RFC1) was markedly higher in rat intestinal villus cells than in crypt cells. These results represent the first identification of a folate transporter in mammalian intestine.

Regulation of intestinal biotin transport in the rat: effect of biotin deficiency and supplementation.

The effect of biotin deficiency and supplementation at pharmacological doses on the intestinal transport of the vitamin was examined in the rat using a brush-border membrane vesicle (BBMV) technique. Transport of biotin in both jejunal and ileal BBMV was significantly (P less than 0.05-0.01) higher in biotin-deficient rats compared with control (pair-fed) rats. This increase in biotin transport appeared to be specific because transport of D-glucose was similar in the two rat groups. The increase in biotin transport in the deficient rats appeared to be mediated through a marked increase (146-230%) in the Vmax of the biotin transport process (with minimal change in the apparent Km), suggesting an increase in the number of the transport carriers. In contrast, supplementation at pharmacological doses of biotin caused significant (P less than 0.05-0.01) and specific decrease (suppression) in biotin transport compared with (unsupplemented) controls. The suppression of biotin transport in the supplemented rats appeared to be mediated through a marked decrease (58%) in the Vmax of the biotin transport process (with minimal change in the apparent Km), suggesting a decrease in the number of the transport carriers. These results provide evidence that biotin transport in the intestine is regulated by the level of the vitamin in the diet (and/or body stores). Furthermore, the results demonstrate the ability of the small intestine to adapt to the challenge of deficiency of an essential nutrient, a capability that may be crucial for the survival of the animal.

Evaluation of a pediatric multiple vitamin preparation for total parenteral nutrition. II. Blood levels of vitamins A, D, and E.

This study represents the first attempt to evaluate the American Medical Association Nutrition Advisory Group (NAG) recommendations for intravenous vitamin A, D, and E dosages for infants and children. Patients studied included 18 preterm infants (group 1) and 26 term infants and children (group 2A) receiving total parenteral nutrition for 2 to 4 weeks and eight infants and children receiving total parenteral nutrition for 3 to 6 months (group 2B). Term gestation infants and children up to 11 years of age all received the same dosages (those that were recommended by the NAG for children weighing more than 10 kg). Preterm infants received 65% of these doses. In group 1, cord blood alpha-tocopherol levels were less than 0.22 mg/dL in seven preterm infants (reference value = 0.29 +/- 0.04), but mean levels increased to 1.65 +/- 0.17 mg/dL after four days of treatment. Eight infants consistently received additional vitamin E orally (80 to 150 mg daily), and their levels increased to 2.18 +/- 0.26 mg/dL by four days of study and to 3.49 +/- 0.57 mg/dL after 3 weeks. Oral supplementation in the preterm infants appeared to be unnecessary because intravenous vitamins alone maintained levels above 1.1 mg/dL. In group 2, alpha-tocopherol levels were maintained within the reference range. Patients receiving lipid emulsions containing substantial quantities of alpha-tocopherol had significantly higher blood levels than patients receiving lipid emulsions containing little alpha-tocopherol (P less than .01). Mean 25-OH vitamin D levels were maintained above or within the reference range in groups 2A and 2B.(ABSTRACT TRUNCATED AT 250 WORDS)

Potential of herbal medicines in modern medical therapy.

The author discusses in this paper the potentialities of Herbal medicine in modern therapy. Also he throws some light on the importance of natural drugs which bring about cure without generation side-effects.