Xue Ping Wang

Role of glutamine depletion in directing tissue-specific nutrient stress responses to L-asparaginase

l-Asparaginase is important in the induction regimen for treating acute lymphoblastic leukemia. Cytotoxic complications are clinically significant problems lacking mechanistic insight. To reveal tissue-specific molecular responses to this drug, mice were administered asparaginase from either Escherichia coli (clinically used) or Wolinella succinogenes (novel, glutaminase-free form). Both enzymes abolished serum asparagine, but only the E. coli form reduced circulating glutamine. E. coli asparaginase reduced protein synthesis in liver and spleen but not pancreas via increased phosphorylation of the translation factor eIF2. In contrast, treatment with Wolinella caused no untoward changes in protein synthesis in any tissue examined. Treating mice deleted for the eIF2 kinase, GCN2, with the E. coli enzyme showed eIF2 phosphorylation to be GCN2-dependent, but only initially. Furthermore, although eIF2 phosphorylation was not increased in the pancreas or by Wolinella asparaginase, expression of the amino acid stress response genes, asparagine synthetase and CHOP/GADD153, increased as a result of both enzymes, even in tissues demonstrating no change in eIF2 phosphorylation. Finally, signaling downstream of the mammalian target of rapamycin kinase was repressed in liver and pancreas by E. coli but not Wolinella asparaginase. These data demonstrate that the nutrient stress response to asparaginase is tissue-specific and exacerbated by glutamine depletion. Importantly, increased expression of asparagine synthetase and CHOP does not require eIF2 phosphorylation, signifying alternate or auxiliary means of inducing gene expression under conditions of amino acid depletion in the whole animal.

Epigenetic Regulation of WTH3 in Primary and Cultured Drug-Resistant Breast Cancer Cells

Previous studies showed that the WTH3 gene functioned as a negative regulator during multidrug resistance (MDR) development in vitro. To understand whether this gene is also involved in clinical drug resistance, hypermethylation at its promoter region observed in cultured MDR MCF7/AdrR cells was examined in primary drug-resistant breast cancer epithelial cells isolated from effusions of breast cancer patients. The results showed that this event also occurred in drug-resistant breast cancer epithelial cells and a newly induced drug-resistant cell line, MCF7/inR. Interestingly, we found that a CpG (CpG 23) that was close to the TATA-like box was constantly methylated in the WTH3 promoter of drug-resistant breast cancer epithelial and cultured MDR cells. Mutagenic study suggested that this CpG site had a functional effect on promoter activity. We also discovered that MCF7/AdrR cells treated with trichostatin A, a histone deacetylase inhibitor, exhibited higher WTH3, but lower MDR1, expression. A reverse correlation between WTH3 and MDR1 gene expression was also observed in MCF7/AdrR, and its non-MDR parental cell line, MCF7/WT. This result indicated that both DNA methylation and histone deacetylase could act in concert to inhibit WTH3 and consequently stimulate MDR1 expression. This hypothesis was supported by data obtained from introducing the WTH3 transgene into MDR cell lines, which reduced endogenous MDR1 expression. Therefore, our studies suggested that the behavior of WTH3 in primary drug-resistant breast cancer epithelial cells was similar to that in a model system where epigenetic regulation of the WTH3 gene was linked to the MDR phenotype.

Automation of the BD GeneOhm Methicillin-Resistant Staphylococcus aureus Assay for High-Throughput Screening of Nasal Swab Specimens

ABSTRACT This study demonstrated that an automated version of the BD-GeneOhm methicillin-resistant Staphylococcus aureus (MRSA) assay (BD-MRSA), using achromopeptidase sample lysis and PCR setup performed on the Hamilton MICROLAB STARlet (Auto-MRSA), gave results comparable to those obtained with BD-MRSA. The positive- and negative-result concordance rates and overall concordance of BD-MRSA and Auto-MRSA were 98.2, 97.7, and 97.6%, respectively. Auto-MRSA required 60% less technical time than BD-MRSA, and the time required to obtain results was 50% of that required for BD-MRSA. Auto-MRSA provides a reliable, robust system for high-volume clinical use.

Mini-bronchoalveolar lavage quantitative polymerase chain reaction for diagnosis of methicillin-resistant Staphylococcus aureus pneumonia

Objectives:Methicillin-resistant Staphylococcus aureus is a common cause of ventilator-associated pneumonia and can be identified by the mecA gene that confers resistance. The objective was to develop a polymerase chain reaction assay for mecA using mini-bronchoalveolar lavage to allow rapid diagnosis of ventilator associated pneumonia attributable to methicillin-resistant Staphylococcus aureus. Design:Real-time quantitative polymerase chain reaction was developed using serial dilution of plasmids containing known amounts of mecA gene fragments. Specificity of molecular identification was based on the presence of the mecA gene and S. aureus-specific femA-SA gene, with absence of Staphylococcus epidermidis specific femA-SE. Setting and Patients:To validate the polymerase chain reaction, we compared it to mini-bronchoalveolar lavage quantitative cultures in 100 mechanically ventilated patients with suspected pneumonia. We derived a threshold value for defining a positive polymerase chain reaction based on a priori criteria of 100% sensitivity. We then tested the polymerase chain reaction using this threshold value on a separate cohort of 50 patients. Main Results:Polymerase chain reaction was able to detect mecA down to a copy number of three, with a high degree of correlation (r2 = .999). The area under the receiver-operating characteristic curve for polymerase chain reaction was 0.98 (95% confidence interval, 0.93–0.99). Using a cut-point of ≥421 for the polymerase chain reaction resulted in sensitivity of 100%, specificity of 87% (95% confidence interval 81%–94%), positive predictive value of 39% (95% confidence interval, 29%–49%), negative predictive value of 100%, and an overall correct classification rate of 89%. Applying the polymerase chain reaction with a cut-point of ≥421 to the second cohort resulted in a sensitivity of 100% and a specificity of 80%. Conclusions:Quantitative polymerase chain reaction of mecA combined with femA-SA and femA-SE detection was able to rapidly and accurately diagnose methicillin-resistant S. aureus.

Correlation of maternal cytophilic human immunodeficiency virus (HIV)-1 V3 loop peptide-specific antibodies in infants with vertical HIV transmission.

ABSTRACT: Approximately 70% of human immunodeficiency virus (HIV)-1-infected pregnant women do not transmit HIV to their offsprings. The identification of factors involved in maternal-child transmission of HIV is important for the design and implementation of therapeutic and prevention strategies. Recently we have developed a modified peripheral mononuclear cell (PBMC) culture system for in vitro antibody production (IVAP) by which we can distinguish serum-derived cytophilic anti-HIV-1 antibody from de novo synthesized antibody. In this study, we analyzed the presence of antibodies directed to HIV-1 gpl60, gpl20, gp41, and V3 loop synthetic peptides (C51, C53, and C57 from MN and HIB strains) utilizing the grid-blot method in PBMC cultures of 52 mother-child pairs. Among the mothers (39 nontransmitters and 13 transmitters), presence of serum-derived cytophilic antibodies or de novo synthesized HIV V3 loop peptide-specific antibodies did not correlate with vertical transmission. However, PBMC-associated, cytophilic antibodies of maternal origin reactive with C51, C53, and C57 from MN and HIB strains were identified in cultures of uninfected infants, but not in infected infants. These observations suggest that cytophilic antibodies of maternal origin directed to HIV-I V3 loop peptides which are bound to infant cells might play a role in preventing vertical transmission.