Biomedical subjects
Publications and source records attributed to H Hamdan.
This is the case of a young woman, 16-year-old, calling to our Department because of the appearance, in the floor of the mouth, of a mass progressively growing until about 5 cm diameter. Through fine needle biopsy was diagnosed of epidermoid cyst. Despite its size and infrahyoid site was removed successfully. Perusal review of last years bibliography and its variants.
Point mutations and inserts in the beta3-beta4 region of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are associated with resistance to nucleoside analog inhibitors. This report describes HIV-1 strains from seven patients that were found to have a 3-bp deletion in the beta3-beta4 region of the RT gene. These patient strains also had a mean of 6.2 drug resistance-associated mutations in their RT genes (range, 3 to 10 mutations). The deletion was most frequently found in strains with the Q151M mutation. Nonnucleoside RT inhibitor mutations were found in six of seven strains. Culture-based drug sensitivity assays showed that deletion-containing isolates had reduced susceptibility to four to eight RT inhibitors. Site-directed mutagenesis experiments showed that the deletion alone conferred reduced susceptibility to nucleoside analogs. Changes in the three-dimensional models of the RT deletion mutants were consistently observed at the beta3-beta4 loop and at helices C and E in both the presence and the absence of dTTP. Loss of hydrogen bonds between the RT and dTTP were also observed in the RT deletion mutant. These results suggest that the deletion in the RT gene contributes to resistance to several nucleoside analogs through a complex interaction with other mutations in the RT gene.
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While many point mutations in the HIV-1 reverse transcriptase (RT) confer resistance to antiretroviral drugs, inserts or deletions in this gene have not been previously characterized. In this report, 14 RT inhibitor-treated patients were found to have HIV-1 strains possessing a 6-basepair insert between codons 69 and 70 of the RT gene. Known drug resistance mutations were also observed in these strains, with T215Y appearing in all strains. Genotypic analysis indicated that the inserts had substantial nucleotide variability that resulted in relatively restricted sets of amino acid sequences. Linkage of patients' treatment histories with longitudinal sequencing data showed that insert strains appeared during drug regimens containing ddI or ddC, with prior or concurrent AZT treatment. Drug susceptibility tests of recombinant patient isolates showed reduced susceptibility to nearly all nucleoside RT inhibitors. Site- directed mutagenesis studies confirmed the role of the inserts alone in conferring reduced susceptibility to most RT inhibitors. The addition of AZT-associated drug resistance mutations further increased the range and magnitude of resistance. These results establish that inserts, like point mutations, are selected in vivo during antiretroviral therapy and provide resistance to multiple nucleoside analogs.
NKX2.1 is a member of the NK2 family of homeodomain-containing transcriptional factors which binds to and activates the promoters of thyroid and pulmonary epithelial genes. We have cloned and sequenced twelve human lung NKx2.1 cDNAs. To elucidate the origin of Nkx2.1 transcripts, we also cloned and sequenced a 12 kb human Nkx2.1 genomic clone. Alignment of cDNA sequences with the genomic clone showed that contrary to previous reports, the human Nkx2.1 gene is organized into three exons and two introns. The newly discovered exon I contains an ATG codon that falls in frame with the previously identified Nkx2.1 initiator ATG codon on one of the cDNAs, designated 5E. Northern blot analysis shows that an mRNA of approximately 2.5 kb in size, homologous to 5E, is expressed in both lung and thyroid. The deduced amino acid sequence of the longest open reading frame on 5E is identical to NKX2.1 with the exception of a 30 amino acid N-terminal extension. Coupled in vitro transcription/translation of the 5E cDNA confirms that the open reading frame is translated into a contiguous polypeptide of 44 kDa. Analysis of Nkx2.1 genomic DNA fragments suggest that at least two independent regions, one within the first intron and the other 5' of the first exon may mediate the basal promoter activity of the Nkx2.1 gene in lung epithelial cells.
Background: The conventional method for diagnosis of fragile X syndrome has been amplification of the trinucleotide repeat region of the FMR-1 gene by polymerase chain reaction (PCR) and Southern blot analysis to detect full expansion and hypermethylation. "Stuttering" resulting from incomplete amplification is still observed in the PCR products despite the use of reagents that reduce the secondary structure of the GC-rich template. In addition, PCR products can be detected by autoradiography only after 1 to 2 days of exposure. By combination of a recently reported amplification protocol with fluorescence detection of PCR products in an automated DNA sequencer, the PCR protocol for amplification of trinucleotide repeats was simplified. This modified protocol is highly reproducible, more accurate, and less costly than the conventional protocol because of the elimination of radioisotopes from the PCR. Methods and Results: PCRs were conducted with betaine and Pfu DNA polymerase. This improved PCR protocol allowed immediate detection of PCR products in agarose gels containing ethidium bromide. Stuttering was completely eliminated and fragments of up to 1kb ( approximately 250 repeats) were visible in agarose gels. PCR products were automatically detected by laser fluorescence in an automated DNA sequencer by inclusion of a fluorescently-labeled primer in the PCR reaction. A short electrophoresis run of 100 minutes in denaturing acrylamide gels was sufficient to give high resolution of fragments with higher accuracy and sensitivity than conventional detection by autoradiography. Conclusions: A simple, nonradioactive protocol that is more rapid and less expensive than the conventional PCR protocol for the detection of trinucleotide repeats has been developed. By use of this detection protocol, fragment sizes containing up to 100 repeats could be detected, alleles differing by one trinucleotide repeat were clearly resolved, and heterogeneous repeat patterns such as those present in mosaics could be discriminated. This protocol has been adapted to the amplification and detection of at least two other classes of trinucleotide repeats [(CAG)(n) and (CTG)(n)], suggesting that it may be a universal protocol for PCR amplification and detection of trinucleotide repeats.
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The effects of exposure to solvents on serum bile acids were investigated by comparing a group of apprentice vehicle spray painters (exposed group) with one of apprentice electricians. Apprentice spray painters from the study were subdivided into high- and low-solvent-exposure groups. Concentrations of individual serum bile acids (SBA) were measured and compared with conventional liver function tests (LFTs). Total, free, glycine- and taurine-conjugated SBA were consistently found to be present at higher levels in the spray painters than in the electricians, even at the beginning of the apprenticeship. Total SBA tended to increase in spray painters with increasing years of exposure during the apprenticeship, but this was significant at only one time point. No rises were observed over the sampling period in electricians. The mean values of individual and total SBA concentrations were all found to be higher in the high-exposure group than in the low-exposure group, with some differences reaching statistical significance. None of the routine liver biochemistry parameters was different between spray painters and electricians. gamma-Glutamyl transferase (GGT) was the only enzyme found to be significantly different between the high- and low-exposure groups, but all values were within the normal range. This study suggests that occupational exposure even to low levels of solvent mixtures results in increases in SBA. The increased SBA may be indicative of a subclinical liver dysfunction. Alternatively, they may reflect solvent exposure only, with the raised levels having no pathologic implication or consequence.
We are interested in determining whether premature birth alters expression of counterregulatory cytokines which modulate lung inflammation. Production of proinflammatory cytokines tumor necrosis factor alpha. IL-1 beta, and IL-8 is regulated in part by the antiinflammatory cytokine IL-10. For preterm newborns with hyaline membrane disease, deficiencies in the ability of lung macrophages to express antiinflammatory cytokines may predispose to chronic lung inflammation. We compared the expression of pro- and antiinflammatory cytokines at the mRNA and protein level in the lungs of preterm and term newborns with acute respiratory failure from hyaline membrane disease or meconium aspiration syndrome. Four sequential bronchoalveolar lavage (BAL) samples were obtained during the first 96 h of life from all patients. All patients rapidly developed an influx of neutrophils and macrophages. Over time, cell populations in both groups became relatively enriched with macrophages. The expression of proinflammatory cytokine mRNA and/or protein was present in all samples from both patient groups. In contrast, IL-10 mRNA was undetectable in most of the cell samples from preterm infants and present in the majority of cell samples from term infants. IL-10 concentrations were undetectable in lavage fluid from preterm infants with higher levels in a few of the BAL samples from term infants. These studies demonstrate that 1) IL-10 mRNA and protein expression by lung inflammatory cells is related to gestational age and 2) during the first 96 h of life neutrophil cell counts and IL-8 expression decrease in BAL from term infants, but remain unchanged in BAL samples from preterm infants.
TTF-1 is a homeodomain transcriptional factor expressed in thyroid, lung, and parts of the brain. In vitro, TTF-1 can activate the promoter of thyroid- and pulmonary-specific genes. We postulated that TTF-1 not only is essential for the activation of tissue-specific genes, but also may directly participate in epithelial cell morphogenesis. To test this postulate, we used an antisense oligonucleotide inhibitory strategy in an in vitro model of embryonic mouse lung branching morphogenesis. This strategy suppressed TTF-1 translation and inhibited lung branching morphogenesis. The resulting abnormal phenotype was characterized by hyperplastic and unorganized proliferation of epithelial cells in the airways. The mesenchymal compartment of the lung appeared to be unaffected. These results demonstrate, for the first time, that the expression of a homeoprotein transcriptional regulator is necessary for lung epithelial morphogenesis.
BACKGROUND: Chemotherapy given by continuous infusion may have different toxicity profiles and different degrees of therapeutic efficacy than when given by bolus administration. The potential therapeutic benefits of continuous infusion chemotherapy and the advantages of outpatient treatment led us to study a continuous infusion of ifosfamide with mesna and oral etoposide. METHODS: The authors performed a Phase I-II trial in which 9 g/m2 ifosfamide was administered for 6 days and 10.5 g/m2 mesna was administered for 7 days, both by continuous infusion, in combination with 50 mg/m2/d oral etoposide for 8 days in 21 patients with sarcomas or other solid tumors. Courses were repeated every 28 days. RESULTS: A total of 65 treatment cycles were given. Only six patients required hospitalization for treatment, all because of an initial poor performance status, and most carried out normal activities on an ambulatory basis. Treatment was stopped during the first course in five patients because of central nervous system toxicity, each with a poor pretreatment performance status; neurologic recovery was complete in each patient. The dose of etoposide was decreased by 20% in 11 patients and unchanged in 7 following the first treatment. Hematologic toxicity was predominantly manifested by leukopenia. An absolute neutrophil count less than 500 neutrophils/microliters occurred in 22 of 50 cycles; thrombocytopenia (platelets less than 100,000/microliters) was seen in two patients, requiring platelet transfusion in one. Neutropenic fevers occurred in 13 of 65 cycles; in 4 of these, cultures demonstrated a bacterial infection. Nausea and vomiting were mild. Objective responses occurred in 6 of 16 patients with soft tissue sarcomas (6 partial responses [PR]) (95% confidence interval, 15-65%), and 3 of 5 bone sarcomas, all of whom had been previously treated with doxorubicin and dacarbazine. CONCLUSIONS: The authors concluded that ifosfamide and mesna given by ambulatory continuous intravenous infusion with wearable pump systems in combination with oral etoposide was well tolerated and showed substantial anti-tumor activity. This combination represents a rational therapeutic approach to patients with advanced soft tissue sarcomas and may have application to other malignancies.
The temporal relationship between trichloroethylene (TRI) and individual serum bile acids (SBA) has been investigated to gain insight into the mechanism of solvent-induced increases in SBA. Male Sprague-Dawley rats were treated with 1 mmol/kg TRI in corn oil, while control rats received only corn oil. Blood samples were collected from the abdominal aorta at 2, 4, 8, and 16 hr after dosing. Individual SBA were determined by high-performance liquid chromatography (HPLC). Liver and blood concentrations of TRI and one of its metabolites, trichloroethanol (TCEOH), were determined by gas chromatography. SBA levels reached their peak at 4 hr and returned to control levels by 16 hr. There was a relationship between SBA levels and TRI concentrations, which were also at their peak 4 hr after dosing. By 16 hr the levels were undetectable. However, peak blood concentrations of TCEOH were reached 8 hr after dosing, and remained high at 16 hr. Cholic acid and taurocholic acid showed the highest levels of bile acids. Some other bile acids were also elevated, including deoxycholic acid, taurodeoxycholic acid, ursodeoxycholic acid, chenodeoxycholic acid, and taurochenodeoxycholic acid. Determination of total bile acids in serum using an enzymatic/colorimetric method showed a similar pattern of response to that obtained with the HPLC analysis. The data are consistent with TRI having a rapid and specific effect on SBA levels by a mechanism other than liver cell damage.
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Pseudomonas fluorescens 2-79 suppresses take-all, a major root disease of wheat caused by Gaeumannomyces graminis var. tritici. The bacteria produce an antibiotic, phenazine-1-carboxylic acid (PCA), and a fluorescent pyoverdin siderophore. Previous studies have established that PCA has an important role in the biological control of take-all but that antibiotic production does not account fully for the suppressiveness of the strain. To define the role of the pyoverdin siderophore more precisely, mutants deficient in production of the antibiotic, the siderophore, or both factors were constructed and compared with the parental strain for control of take-all on wheat roots. In all cases, strains that produced PCA were more suppressive than those that did not, and pyoverdin-deficient mutant derivatives controlled take-all as effectively as their respective fluorescent parental strains. Thus, the phenazine antibiotic was the dominant factor in disease suppression and the fluorescent siderophore had little or no role. The siderophore also was of minor importance in a second strain, P. fluorescens M4-80R, that does not produce PCA. Strains 2-79 and M4-80R both produced substances distinct from the pyoverdin siderophore that were responsible for fungal inhibition in vitro under iron limitation, but these substances also had, at most, a minor role in disease suppression in situ.