The EbpA-RpoN Regulatory Pathway of the Pathogen Leptospira interrogans Is Essential for Survival in the Environment.

Leptospira interrogans is the agent of leptospirosis, a reemerging zoonotic disease. It is transmitted to humans through environmental surface waters contaminated by the urine of mammals chronically infected by pathogenic strains able to survive in water for long periods. Little is known about the regulatory pathways underlying environmental sensing and host adaptation of L. interrogans during its enzootic cycle. This study identifies the EbpA-RpoN regulatory pathway in L. interrogans In this pathway, EbpA, a σ54 activator and putative prokaryotic enhancer-binding protein (EBP), and the alternative sigma factor RpoN (σ54) control expression of at least three genes, encoding AmtB (an ammonium transport protein) and two proteins of unknown function. Electrophoresis mobility shift assay demonstrated that recombinant RpoN and EbpA bind to the promoter region and upstream of these three identified genes, respectively. Genetic disruption of ebpA in L. interrogans serovar Manilae virtually abolished expression of the three genes, including amtB in two independent ebpA mutants. Complementation of the ebpA mutant restored expression of these genes. Intraperitoneal inoculation of gerbils with the ebpA mutant did not affect mortality. However, the ebpA mutant had decreased cell length in vitro and had a significantly lowered cell density at stationary phase when grown with l-alanine as the sole nitrogen source. Furthermore, the ebpA mutant has dramatically reduced long-term survival ability in water. Together, these studies identify a regulatory pathway, the EbpA-RpoN pathway, that plays an important role in the zoonotic cycle of L. interrogans IMPORTANCE: Leptospirosis is a reemerging disease with global importance. However, our understanding of gene regulation of the spirochetal pathogen Leptospira interrogans is still in its infancy, largely due to the lack of robust tools for genetic manipulation of this spirochete. Little is known about how the pathogen achieves its long-term survival in the aquatic environment. By utilizing bioinformatic, genetic, and biochemical methods, we discovered a regulatory pathway in L. interrogans, the EbpA-RpoN pathway, and demonstrated that this pathway plays an important role in environmental survival of this pathogen.

[Study on HPLC Fingerprint of Trichosanthes Fruit and Its Processed Products].

OBJECTIVE: To study the correlation on chemical fingerprint features between Trichosanthis Fructus and its processed products. METHODS: The chemical fingerprints were established by HPLC for the ethyl acetate extraction and the n-butanol extraction in Trichosanthis Fructus and its processed products,the common pattern was established by the mean and the median, and the similarity degree between Trichosanthis Fructus and its processed products was calculated by the correlation coefficient method and the included angle cosine method. RESULTS: There were 24 common peaks in the fingerprints of ethyl acetate extraction of Trichosanthis Fructus and its processed products,the average similarity degree was calculated separately by the correlation coefficient method and the included angle cosine method:the former as the value was 0. 8497(mean), 0. 8344(median); and the latter as the value was 0. 8429(mean), 0. 8536 (median); there were 6 common peaks in the fingeprints of n-butanol extraction of Trichosanthis Fructus and its processed products, the average similarity degree was calculated by the correlation coefficient method and the included angle cosine method:the former as the value was 0. 9044 (mean), 0. 9076 (median); and the latter as the value was 0. 9075 (mean), 0. 9081 (median). CONCLUSION: Main peaks have good correlation between Trichosanthis Fructus and its processed products. This method can provide theoretical basis for processing and quality control of Trichosanthis Fructus.

[Spectrum-Effect Relationship of GualouXiebai Dropping Pills on Myocardial Ischemia].

OBJECTIVE: To study the relationship between HPLC characteristic spectrum and pharmacodynamics on anti-myocardial ischemia of GualouXiebai dropping pills. METHODS: HPLC characteristic spectrum of GualouXiebai dropping pills was established, dropping pills were divided into five dose groups (3.75, 11.25, 22.5, 33.75 and 45 g/kg, equivalent to the crude herb g/kg), the mice were orally administered dropping pills once daily for 7 d, 90 min after the mice were given by intraperitoneal injection of isoprenaline to establish myocardial ischemia models, the level of CK in blood plasma were detected; Then, the correlation between characteristic spectrum and biochemical index CK was studied by grey relational analysis method. RESULTS: The correlation between each common peak and CK had gradually increased with the dose increased from 3.73 g/kg to 33.75 g/kg, but when the dose reached to 45 g/kg, the correlation between each common peak and CK had decreased. The variation trends of correlation of spectrum-effect relationship for different dose were similar,but the correlation variation trend of the efficacy on the No. 8 peak in 33.75 g/kg group with the other four groups in the opposite, the change trends of the No. 11 peak in 22.5 g/kg group, the No. 24 peak in 33. 75 g/kg group and the No. 37 peak in 45 g/ kg group with 3.75 g/kg group and 11.25 g/kg group on the contrary. The relational orders of spectrum-effect relationship were not consistent, respectively( the first 15 peaks) :11 > 37 > 24 > 30 > 8 > 21 > 2 > 16 > 1 > 3 > 20 > 15 > 12 > 19 > 7;11 > 37 > 30 > 8 > 21 > 24 > 2 > 1 > 16 > 3 > 27 > 12 > 22 > 20 >10; 8 > 30 > 1 > 2 > 21 > 27 > 31 > 22 > 16 > 12 > 3 > 10 > 9 > 20 > 4; 1 > 2 > 27 > 21 > 31 > 22 > 12 > 16 > 9 > 3 > 10 > 4 > 17 > 30 > 20; 8 > 30 > 1 > 2 > 2 > 2 > 7 > 31 > 22 > 16 > 12 > 3 > 9 > 10 > 20 > 17. CONCLUSION: Anti-myocardial ischemia effect of GualouXiebai dropping pills comes from the synergistic or antagonistic effect among various active ingredients related to the dose. With the difference of the dosage, the relational orders of chemical components to play the role is not the same, but the main components to play a pharmacodynamic of five dose groups are consistent,the existence of the component groups lay a foundation for further study of GualouXiebai dropping pills.

Structure and Function of a Novel Silencer in 5' Flanking Distal Region of Human beta Globin Gene.

A novel silencer fragment(310 bp) was recently discovered and identified to locate between -2 132 bp and -1 822 bp upstream from the cap site of beta-globin gene by gel retardation assay and luciferase reporter gene expression analysis. DNA footprinting assays were performed to determine the interaction between its DNA sequence and binding proteins from the nuclear extract of Hela cells. The results showed that there were two nuclear protein binding sites in thissilencer, one was the -2 017--2 011 bp sequence CTTCCGC" and the other was the -2 006--1 997 bp sequence "CACTTTATTT". Two sequences were mutated into "CTTAAGC" and "CACTTAAGTT", respectively by two mutagenic primer pairs, in order to construct two mutation types of the 310 bp fragment. The competitive gel retardation assays showed that two mutation types of the 310 bp fragment and their four smaller DNA fragments, which were formed respectively by the digestion of restriction enzyme BspTI, all lost their competitive ability against the wild type of 310 bp fragment probe for DNA-binding proteins without exception. Furthermore, the double-strand oligonucleotides, which contained both the sequences of "CTTCCGC" and "CACTTTATTT", were synthesized, and the competitive gel retardation assays showed that they competed ability against wild type 310 bp fragment probe for DNA-binding proteins. The results suggest that two binding sites of the nuclear proteins are involved or associated with a potential DNA-DNA interaction. Moreover, the specific DNA-binding proteins were purified from the nuclear extract of Hela cells by using "DNA-binding protein purification kit" for magnetic isolation. In order to identify the purified DNA-binding proteins, a SDS-PAGE was performed. By using the silver staining, the PAGE electrophoretogram showed that these two nuclear proteins specifically bound to these two sites of the silencer, appearing as two definite bands. The molecular weight of each protein was determined to be 37 kD or 81 kD, respectively.

Function and Identification of a Novel Silencer in 5' Flanking Distal Region of Human beta Globin Gene.

In order to inquire into the possible function of the sequence in the 5' flanking distal region and/or delta-beta intergene of human beta-globin gene several recombinant retrovirus vectors containing beta-globin gene with different length of 5' flanking sequences were constructed and transfected into Hela cells. The levels of beta-globin gene expression determined quantitatively by Northern blotting and RT-cPCR revealed that there is a strong silencer activity in a DNA sequence(723 bp) between-2282 bp and -1559 bp upstream from the cap site of beta-globin gene. The silencer activity was further identified to be possibly located mainly on a 310bp DNA restriction fragment by using the competitive gel retardation assay(cGRA) and many known regulatory elements were found present in it as well. Luciferase reporter gene expression system was adopted to study the function of this 310bp fragment and data showed that the fragment exhibited significant inhibitory effect on each of the recombinant vectors with alpha,beta or delta gene promoters. The above results suggest that this silencer may be involved in a potential regulatory network between delta and beta globin genes and may play an important role in the regulation of globin gene expression.