In vitro activity of natural phenolic compounds against fluconazole-resistant Candida species: a quantitative structure-activity relationship analysis.

AIMS: To evaluate the antifungal activity and to analyse the structure-activity relationship of eleven natural phenolic compounds against four Candida species which are resistant to fluconazole. METHODS AND RESULTS: Four different species of Candida isolates were used: Candida albicans, Candida krusei, Candida tropicalis and Candida dubliniensis. The phenolic compound carvacrol showed the highest anti-Candida bioactivity, followed by thymol and isoeugenol. The obtained minimum inhibitory concentration (MIC) values obtained were used in a quantitative structure-activity relationship (QSAR) analysis where the electronic, steric, thermodynamic and topological descriptors served as dependent variables. According to the descriptors obtained in this QSAR study, the antifungal activity of phenols has a first action specific character which is based on their interaction with plasma or mitochondrial membranes. The second action is based on a steric descriptor-the maximal and minimal projection of the area-which could explain the inability of some phenolic compounds to be biotransformed to quinones methylene by Candida species. CONCLUSIONS: According to the descriptors obtained in this QSAR study, the anti-Candida activity of ortho-substituted phenols is due to more than one action mechanism. The anti-Candida activity of phenolic compounds can be predicted by their molecular properties and structural characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: These results could be employed to predict the anti-Candida activity of new phenolic compounds in the search for new alternatives or complementary therapies to combat against candidiasis.

Chemical constituents and antimicrobial activity of the essential oil of Lantana xenica.

The aim of this work was to evaluate the chemical composition of Lantana xenica essential oil and its antimicrobial activity. The oil from the aerial parts of Lantana xenica Mold. (Verbenacea) was obtained by steam distillation and analyzed by gas chromatography/mass spectrometry. The major constituent of the oil was (E)-caryophyllene (35.2 %), with minor amounts of gamma-cadinene (13.3 %), alpha-pinene (9.3 %), ocimene (9.2 %) and germacrene D (6.6 %). The antimicrobial assays showed that the essential oil of L. xenica inhibited the growth of Bacillus cereus and Proteus mirabilis and both bacteria were inhibited by (E)-caryophyllene, the major component of the oil. Enterococcus faecalis, Staphylococcus epidermidis and S. aureus showed a lower inhibition. The bacteria Micrococcus luteus, Klebsiella sp., Escherichia coli and the yeast Candida albicans were insensitive to both the oil and (E)-caryophyllene.

Colonic epithelium-enriched protein A4 is a proteolipid that exhibits ion channel characteristics.

Expression of the human gene A4 is enriched in the colonic epithelium and is transcriptionally activated on differentiation of colonic epithelial cells in vitro (M. M. Oliva, T. C. Wu, and V. W. Yang. Arch. Biochem. Biophys. 302: 183-192, 1993). A4 cDNA contains an open reading frame that predicts a polypeptide of 17 kDa. To determine the function of the A4 protein, we characterized its biochemical and physiological properties. Hydropathy analysis of deduced A4 amino acid sequence revealed four putative membrane-spanning alpha-helices. The hydrophobic nature of A4 was confirmed by its being extractable with organic solvents. Immunocytochemical studies of cells expressing A4 localized it to the endoplasmic reticulum. Moreover, A4 multimerized in vivo as determined by coimmunoprecipitation experiments. The four-transmembrane topology and biophysical characteristics of A4 suggest that it belongs to a family of integral membrane proteins called proteolipids, some of which multimerize to form ion channels. Subsequent electrophysiological studies of nuclei isolated from microinjected Xenopus laevis oocytes transiently expressing A4 showed the appearance of a 28-pS channel. Thus our studies indicate that A4 is a colonic epithelium-enriched protein localized to the endoplasmic reticulum and that, similar to other proteolipids, A4 multimerizes and exhibits characteristics of an ion channel.

Skull infarction and epidural hematomas in a patient with sickle cell anemia.

PURPOSE: Epidural hematomas are unusual manifestations of sickling disorders. We report a patient with sickle cell anemia and multiple skull infarctions associated with epidural hematomas. The association of skull infarctions and epidural hematomas in sickling hemoglobinopathies is reviewed. PATIENTS AND METHODS: A 14-year-old boy with hemoglobin SS presented with lower back pain, left hip pain, headache, and fever. A bone scan was used to evaluate the patient for possible osteomyelitis. Head computed tomography (CT) and magnetic resonance imaging (MRI) were employed to delineate intracranial pathology. RESULTS: The bone scan showed multiple areas of decreased uptake in the skull consistent with acute infarction before abnormalities were present on physical examination. CT scan showed a bony contour deformity of the right frontal bone, suggestive of infarction. A right frontal extra-axial collection of blood was also observed below the bony abnormality. MRI further delineated bilateral frontal and left parietal collections adjacent to the bony abnormalities consistent with subacute epidural hematomas. CONCLUSIONS: This case emphasizes the need to recognize skull infarctions and epidural hematomas as rare but potential complications of sickle cell disease. The diagnosis was facilitated by MRI, which has not been used in previous cases of skull infarctions. Moreover, our patient was successfully managed without surgical intervention.

Nutritional considerations and management of the child with inflammatory bowel disease.

Crohn's disease and ulcerative colitis are chronic inflammatory diseases of the bowel often associated with significant malnutrition, particularly in children because of increased nutrient demands due to growth. We discuss the increasingly prominent role of nutritional support in inflammatory bowel disease (IBD). Issues that are addressed include the etiology of malnutrition in IBD, assessment and monitoring of patient nutritional status and the use of nutrition in the management of growth failure and as primary medical therapy.

Comparison of local injection of methotrexate and linear salpingostomy in the conservative laparoscopic treatment of ectopic pregnancy.

STUDY OBJECTIVE: To compare local injection of metothrexate (MTX) and linear salpingostomy in the conservative laparoscopic treatment of ectopic pregnancy. DESIGN: Prospective, nonrandomized study, July 1991 to May 1994. SETTING: Department of obstetrics and gynecology in a university hospital. PATIENTS: Fourteen women with unruptured ectopic pregnancies without documented fetal heart motion and size below 50 mm as measured by ultrasound. INTERVENTIONS: All 14 women underwent the laparoscopic treatment by either local injection of MTX or linear salpingostomy (7 patients each). MEASUREMENTS AND MAIN RESULTS: Both treatments were successful in all patients. Mean length of operation was 32 +/- 5 minutes (range 25-35 min) in the MTX group versus 67 +/- 15 minutes (range 50-90 min) in the salpingostomy group. Mean length of hospital stay was 2.7 days (range 1-5 days) and 1.7 days (range 1-3 days), respectively. No intraoperative complications occurred, and the postoperative course was uneventful in all women. Mean disappearance time of serum beta-human chorionic gonadotropin (hCG) levels was similar in both groups, although in the linear salpingostomy group the decrease was immediate. No difference in tubal patency on follow-up hysterosalpingography was observed between the two groups. CONCLUSIONS: Although this is a preliminary report with a small number of patients, both types of treatment were safe and effective. An advantage of linear salpingostomy was the predictable and consistent decline of circulating beta-hCG, and consequently a reduced need for a close follow-up. Local MTX injection was safe, economic, effective, and easy to perform, and in our experience the surgical time was statistically shorter than that for linear salpingostomy. Therefore, in selected patients, local injection of MTX could be the treatment of choice for unruptured ectopic pregnancy, avoiding a longer and potentially more dangerous procedure. Long-term outcomes do not seem to differ between the two types of treatment.

Promoter regulation of a differentially expressed gene in the human colonic epithelial cell lines HT29-18 and HT29-18-C1.

Gene A4 is transcriptionally activated upon enterocyte differentiation of the human colonic epithelial cell line HT29-18 and its highly differentiated subclone HT29-18-C1 [Oliva et al., Arch. Biochem. Biophys. 302 (1993) 183-192]. To characterize the mechanisms regulating the differential transcription of A4, we analyzed its immediate 5'-flanking region for regulatory elements. Promoter-linked transfection experiments of progressively deleted A4 5'-flanking sequences fused to the bacterial cat reporter gene suggest the presence of one negative and two positive DNA elements within the first 371 bp of the A4 promoter (pA4). DNase I footprint and electrophoretic mobility shift assays demonstrate that one positive element which contains the core binding sequence for the transcription factor, Sp1, mediates an equal level of transcription in the two cell types. The second positive element, localized between nucleotide positions--169 and -152, contains a sequence previously unrecognized as a transcription factor-binding site. This element mediates a twofold increase in the activity of pA4 in HT29-18-C1, as compared to HT29-18. Furthermore, nuclear extracts obtained from HT29-18-C1 contain a higher binding activity for this element than those from HT29-18. Southwestern blot analysis suggests that the protein interacting with this element has an estimated molecular mass of 50 kDa. We conclude that this protein may be involved in the differential regulation of A4 in these intestinal cell lines.

Successful intestinal transplantation for microvillus inclusion disease.

Microvillus inclusion disease is a rare congenital disorder that presents with severe diarrhea in the newborn period. Multiple therapeutic attempts to control the diarrhea have failed, leading to a chronic dependence on parenteral nutrition and a high infant mortality. This report presents the first child with microvillus inclusion disease in whom small bowel transplantation has been successful, allowing for the administration of total caloric requirements via the enteral route.

Gastritis associated with Gastrospirillum hominis in children. Comparison with Helicobacter pylori and review of the literature.

Interest in possible microbiological causes of gastritis has significantly increased since the discovery of Helicobacter pylori. Recently a spiral bacterium named Gastrospirillum hominis was described in association with chronic gastritis in adult patients. Here, we present the finding of Gastrospirillum hominis in the gastric biopsies of two children who underwent upper endoscopy for gastrointestinal symptoms. The frequency of Gastrospirillum hominis (0.3%) in our pediatric population was similar to that reported in adults. We observed a chronic gastritis associated with the spiral bacteria which was milder than the gastritis noted in our pediatric patients with Helicobacter pylori infection. Further comparisons between these two organisms, as well as the literature on Gastrospirillum hominis, are also reviewed.

Isolation and characterization of a differentiation-dependent gene in the human colonic cell line HT29-18.

The human colonic epithelial cell line HT29, and its clonal derivatives HT29-18 and HT-29-18-C1, differentiate in vitro. Differential screening of a subtraction cDNA library enriched for sequences unique to HT29-18-C1, a highly differentiated subclone of HT29-18, resulted in the isolation of a differentiation-dependent cDNA clone, A4. A full-length clone encoding A4 was obtained and sequenced to its entirety. It is 945 bp in length and contains an open reading frame (ORF) of 456 bp. The amino acid sequence deduced from the ORF reveals a polypeptide of 152 amino acids with a predicted molecular mass of 17,000 Da, a size confirmed by coupled in vitro transcription and translation directed by the full-length A4 cDNA. This polypeptide contains four potential membrane-spanning domains and consensus sequences for N-linked glycosylation as well as phosphorylation sites for protein kinase C and casein kinase II. Comparison of A4 to published DNA and protein sequences revealed no significant homology. Genomic Southern blot analysis suggests that the gene is present in a single copy within the human genome and is conserved in the rat. Northern blot analysis of RNA obtained from various rat tissues shows that the expression of the A4 gene is tissue-selective and is enriched in colonic mucosa. In situ hybridization using human intestinal tissues indicates that the expression of A4 follows a gradient along the crypt-to-villus axis with the most abundant message occurring in the lower half of the crypt. Furthermore, nuclear run-on assays suggest that the induction of the A4 gene during differentiation of HT29-18 is regulated at a transcriptional level. A clone was isolated from a human genomic library and found to contain all five exons of A4. S1 nuclease analysis localized the start site of transcription to an adenosine residue 91 nucleotides upstream from the ATG translation initiation codon. Examination of the immediate sequence 5' to the mRNA start site reveals no TATA box and multiple known enhancer sequences. A4 is also noted to share certain features with the gene encoding the cystic fibrosis transmembrane conductance regulator protein. They include a similar vertical distribution of expression along the intestinal epithelium, enhanced transcription upon differentiation of HT29-18, and multiple shared putative regulatory sequences in the promoter regions. Further characterization of the mechanisms regulating expression of the A4 gene could contribute to the understanding of mammalian intestinal differentiation.

Enhancer-site downstream binding protein activity is enriched in rat tissues that express the class I alcohol dehydrogenase gene.

The activity of the rat class I alcohol dehydrogenase (ADH) is enriched in certain tissues including the liver, intestine and testis. The tissue-specific expression of the gene encoding ADH in the rat was studied and found to closely correlate with tissue isozymic activity. A factor designated enhancer-site downstream binding protein (EDBP) was recently identified in the rat liver and found to interact with the proximal promoter of the class I ADH gene. The distribution of EDBP in nuclear extracts obtained from various tissues was examined based on its sequence-specific DNA binding property and found to correlate with tissue ADH expression. These findings suggest that EDBP is potentially a positive regulatory factor which is involved in controlling the tissue-specific expression of the ADH gene.

Regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gene transcription and alternative RNA splicing in a model of developing intestinal epithelium.

Transcriptional and post-transcriptional regulation of CFTR (cystic fibrosis transmembrane conductance regulator) gene expression was studied in HT29 cells. It is known that the abundance of CFTR mRNA increases during differentiation of pluripotent HT29-18 cells and is maintained at high levels in the stably differentiated HT29-18-C1 subclone. Nuclear run-on assays suggest that increased transcription of the CFTR gene explains the increased abundance of total CFTR mRNA in differentiated HT29 cells. The increased transcription cannot be ascribed to cell cycle-dependent expression of the CFTR gene or to changes in CFTR gene copy number between subcloned cells. Similar to native tissue cells, differentiated HT29 cells contain low copy numbers of CFTR transcripts (1-5/cell), and a portion of the CFTR transcripts are alternatively spliced to remove exon 9 (and make 9-mRNA). During differentiation of HT29-18 cells, the absolute amount of full-length CFTR mRNA increases 8-fold, whereas the amount of 9- mRNA increases 18-fold. The fraction of 9- mRNA in the CFTR mRNA pool is increased in differentiated HT29 cells. The results show that gene transcription regulates the abundance of CFTR transcripts and that regulatory control of alternative RNA splicing may also be a cellular mechanism to modulate CFTR function.