Conjugative transfer, stability and expression of a plasmid encoding a cry1Ac gene in Bacillus cereus group strains

The plasmid pHT73 containing cry1Ac and tagged with an erythromycin resistance gene was transferred from Bacillus thuringiensis subspecies kurstaki KT0 to several Bacillus cereus group strains by conjugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and phase contrast microscopy showed that the transconjugants containing plasmid pHT73 could express Cry1Ac toxin and produce bipyramidal crystalline inclusion bodies during sporulation. The study demonstrated that pHT73 could be transferred to B. thuringiensis subsp. kurstaki, several B. cereus strains and Bacillus mycoides. Under non-selective conditions, the stability of the pHT73 plasmid in the transconjugants was found to be 58.2-100% after 100 generations and 4-96% after 200 generations. The variations are mainly caused by the choice of receptor strain.     

Determination of vincristine in mouse plasma and brain tissues by liquid chromatography-electrospray mass spectrometry

Vincristine is an anticancer agent that continues to be examined in preclinical models even though it is used in a variety of human neoplastic disorders. We developed a sensitive liquid chromatography-mass spectrometry (LC-MS) method for the determination of vincristine in plasma and in brain tissues that would support investigations on drug distribution into tissues in animal models. The procedure required only a small sample volume (10 microl) of plasma, which circumvented a limitation of most other assays that were developed for human samples. A solid-phase extraction procedure was employed that enabled the eluent to be directly injected onto a reversed-phase chromatographic HPLC system using positive electrospray ionization followed by mass spectrometric detection. The extraction recoveries of vincristine were 57 and 60% from plasma and brain tissues, respectively. The mobile phase consisted of methanol and 15 mM ammonium acetate in 0.02% formic acid (70:30) that was pumped at 0.2 ml/min to yield retention times of 1.6 and 1.8 min for vincristine and vinblastine, the internal standard, respectively. The method was validated at vincristine plasma concentrations from 0.01 to 2 microg/ml, and from 0.01 to 1 microg/g in brain tissue. The advantage of the method enabled the quantitation of vincristine in multiple plasma samples obtained from a single mouse, which permitted the accurate estimation of its pharmacokinetic properties.     

Inhibitory mode of indole-2-carboxamide derivatives against HLGPa: molecular docking and 3D-QSAR analyses

The interaction of a series of indole-2-carboxamide compounds with human liver glycogen phosphorylase a (HLGPa) have been studied employing molecular docking and 3D-QSAR approaches. The Lamarckian Genetic Algorithm (LGA) of AutoDock 3.0 was employed to locate the binding orientations and conformations of the inhibitors interacting with HLGPa. The binding models were demonstrated in the aspects of inhibitor's conformation, subsite interaction, and hydrogen bonding. The very similar binding conformations of these inhibitors show that they interact with HLGPa in a very similar way. Good correlations between the calculated interaction free energies and experimental inhibitory activities suggest that the binding conformations of these inhibitors are reasonable. The structural and energetic differences in inhibitory potencies of indole-2-carboxamide compounds were reasonably explored. Using the binding conformations of indole-2-carboxamides, consistent and highly predictive 3D-QSAR models were developed by CoMFA and CoMSIA analyses. The q2 values are 0.697 and 0.622 for CoMFA and CoMSIA models, respectively. The predictive ability of these models was validated by four compounds that were not included in the training set. Mapping these models back to the topology of the active site of HLGPa leads to a better understanding of the vital indole-2-carboxamide-HLGPa interactions. Structure-based investigations and the final 3D-QSAR results provide clear guidelines and accurate activity predictions for novel inhibitor design.     

Mutational inactivation of two distinct negative RNA elements in the human papillomavirus type 16 L2 coding region induces production of high levels of L2 in human cells

Here we show that the 5' end and the middle region of the L2 coding sequence of human papillomavirus type 16 contain strong inhibitory RNA sequences termed inhibitory regions I and II. This is in contrast to L1, which contains one inhibitory region in the 5' end of the coding region. Inhibitory regions I and II acted in cis to reduce L2 mRNA levels and to inhibit the use of the mRNA. In tandem, the two regions reduced L2 mRNA production to undetectable levels. Specific mutational inactivation of the two inhibitory elements in the 5' end and in the middle region of L2 by the introduction of nucleotide substitutions that changed the nucleotide sequence but not the protein sequence resulted in production of high levels of L2 mRNA and protein. In contrast to L2, a partial L1 mutant in which only the first one third of L1 was mutated produced levels of L1 mRNA and protein similar to those in a full L1 mutant. In addition, the constitutive transport element of simian retrovirus type 1 overcomes the effect of the inhibitory sequences of L1 but not L2.     

PCR和分离培养同时检测儿童呼吸道感染的支原体

为了解广州市儿童呼吸道支原体感染情况,用一条共同的上游引物,二条特异性的下游引物建立的ＰＣＲ方法能同时扩增ＭＰ的６９１ｂｐ和ＭＧ的４３８ｂｐ粘附因子基因片段,但不会扩增其他支原体和细菌的ＤＮＡ。     

Scalable Production in Human Cells and Biochemical Characterization of Full-Length Normal and Mutant Huntingtin

Huntingtin (Htt) is a 350 kD intracellular protein, ubiquitously expressed and mainly localized in the cytoplasm. Huntington’s disease (HD) is caused by a CAG triplet amplification in exon 1 of the corresponding gene resulting in a polyglutamine (polyQ) expansion at the N-terminus of Htt. Production of full-length Htt has been difficult in the past and so far a scalable system or process has not been established for recombinant production of Htt in human cells. The ability to produce Htt in milligram quantities would be a prerequisite for many biochemical and biophysical studies aiming in a better understanding of Htt function under physiological conditions and in case of mutation and disease. For scalable production of full-length normal (17Q) and mutant (46Q and 128Q) Htt we have established two different systems, the first based on doxycycline-inducible Htt expression in stable cell lines, the second on “gutless” adenovirus mediated gene transfer. Purified material has then been used for biochemical characterization of full-length Htt. Posttranslational modifications (PTMs) were determined and several new phosphorylation sites were identified. Nearly all PTMs in full-length Htt localized to areas outside of predicted alpha-solenoid protein regions. In all detected N-terminal peptides methionine as the first amino acid was missing and the second, alanine, was found to be acetylated. Differences in secondary structure between normal and mutant Htt, a helix-rich protein, were not observed in our study. Purified Htt tends to form dimers and higher order oligomers, thus resembling the situation observed with N-terminal fragments, although the mechanism of oligomer formation may be different.     

Lactate Dehydrogenase B Is Associated with the Response to Neoadjuvant Chemotherapy in Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma (OSCC) comprises a subset of head and neck squamous cell carcinoma (HNSCC) with poor therapeutic outcomes and high glycolytic dependency. Neoadjuvant chemotherapy regimens of docetaxel, cisplatin and 5-fluorouracil (TPF) are currently accepted as standard regimens for HNSCC patients with a high risk of distant metastatic spread. However, the antitumor outcomes of TPF neoadjuvant chemotherapy in HNSCC remain controversial. This study investigated the role of lactate dehydrogenase B (LDHB), a key glycolytic enzyme catalyzing the inter-conversion between pyruvate and lactate, in determining chemotherapy response and prognosis in OSCC patients. We discovered that a high protein level of LDHB in OSCC patients was associated with a poor response to TPF regimen chemotherapy as well as poor overall survival and disease-free survival. Our in-depth study revealed that high LDHB expression conferred resistance to taxol but not 5-fluorouracil or cisplatin. LDHB deletion sensitized OSCC cell lines to taxol, whereas the introduction of LDHB decreased sensitivity to taxol treatment. Taxol induced a pronounced impact on LDHB-down-regulated OSCC cells in terms of apoptosis, G2/M phase cell cycle arrest and energy metabolism. In conclusion, our study highlighted the critical role of LDHB in OSCC and proposed that LDHB could be used as a biomarker for the stratification of patients for TPF neoadjuvant chemotherapy and the determination of prognosis in OSCC patients.     

Association of Urinary Metal Profiles with Altered Glucose Levels and Diabetes Risk: A Population-Based Study in China

Background

Elevated heavy metals and fasting plasma glucose (FPG) levels were both associated with increased risk of cardiovascular diseases. However, studies on the associations of heavy metals and essential elements with altered FPG and diabetes risk were limited or conflicting. The objective of this study was to evaluate the potential associations of heavy metals and essential trace elements with FPG and diabetes risk among general Chinese population.

Methods

We conducted a cross-sectional study to investigate the associations of urinary concentrations of 23 metals with FPG, impaired fasting glucose (IFG) and diabetes among 2242 community-based Chinese adults in Wuhan. We used the false discovery rate (FDR) method to correct for multiple hypothesis tests.

Results

After adjusting for potential confounders, urinary aluminum, titanium, cobalt, nickel, copper, zinc, selenium, rubidium, strontium, molybdenum, cadmium, antimony, barium, tungsten and lead were associated with altered FPG, IFG or diabetes risk (all P< 0.05); arsenic was only dose-dependently related to diabetes (P< 0.05). After additional adjustment for multiple testing, titanium, copper, zinc, selenium, rubidium, tungsten and lead were still significantly associated with one or more outcomes (all FDR-adjusted P< 0.05).

Conclusions

Our results suggest that multiple metals in urine are associated with FPG, IFG or diabetes risk. Because the cross-sectional design precludes inferences about causality, further prospective studies are warranted to validate our findings.