工程菌DH5α/pCW-PL-XE-TNFαm2的遗传稳定性

目的本基因工程大肠杆菌DH5α/pCW-PL-XE-TNFαm2所表达的靶向融合蛋白XE-TNFαm2已被初步证明具有用于清除艾滋病患者体内HIV病毒的前景。其目的蛋白表达水平为32%～36%细胞总蛋白。本研究旨在验证其遗传稳定性。方法工程菌株DH5α/pCW-PL-XE-TNFαm分别在LBAmp＋与LBAmp-二种固体培养基上逐日单菌落划线传代,32℃培养过夜。每间隔十代运用一般温控表达技术,确定其XE-TNFαm2的蛋白含量,最后比较分析各代之间目的蛋白（20.3 kDa）表达水平的差异情况。结果该重组基因工程菌连续传100代后XE-TNFαm2的蛋白表达水平没有明显差异（P〉0.05）;只是在上述两种情况下传至100代后将其置于4℃保藏4、5、6个月,其目的蛋白表达水平有8%的下降。本载体质粒含有的CIts857序列、PL启动子与T1T2末端终止序列,是确保目的基因稳定高表达的3个关键元件。结论本研究结果证明该工程菌DH5α/pCW-PL-XE-TNFαm2具有良好的遗传稳定性。     

赤藓糖醇对主要致龋链球菌及耐氟菌株生长和产酸的影响

目的通过赤藓糖醇对变形链球菌、远缘链球菌及其耐氟菌株混合菌生长和产酸影响的体外研究,为赤藓糖醇防龋作用的机理提供制论依据。方法采用最小抑菌浓度递增法对变形链球菌（S.mutans ATCC 25175,S.m）、远缘链球菌（S.sobrinus 6715,S.s）进行氟化钠体外诱导耐氟菌株（S.m-FR、S.s-FR）,利用液体稀释法配制赤藓糖醇TSB液8个浓度,分别加入含有变形链球菌、远缘链球菌及其耐氟菌株的细菌混悬液48 h,用比浊法观察其对混合菌生长的影响,并用pH计测定培养前后上清液的△pH值。结果吸光度A值和△pH值实验前后与对照组相比最低浓度为12%时差异均有统计学意义（P〈0.05）,且随着浓度的升高A值和△pH值均下降。结论赤藓糖醇能抑制变形链球菌、远缘链球菌及耐氟菌株混合菌生长和产酸,并且随着浓度的升高抑制作用增强。     

米诺环素对泛耐药鲍曼不动杆菌的体外药敏分析

初步探讨米诺环素对泛耐药的鲍曼不动杆菌体外药敏。将各种标本分离获得的鲍曼不动杆菌,进行米诺环素药敏试验,并将检测结果进行统计和分析。2009年1月至2011年9月共检出鲍曼不动杆菌471株,其中米诺环素敏感的鲍曼不动杆菌2009年5株、2010年12株、2011年23株;亚胺培南、美洛培南敏感其他耐药的0株,亚胺培南、美洛培南、米诺环素敏感其他耐药的0株;米诺环素和头孢哌酮/舒巴坦同时敏感的236株,占总数的50.1%。米诺环素对泛耐药的鲍曼不动杆菌在体外有很好的抗菌活性,特别是联合头孢哌酮/舒巴坦治疗泛耐株鲍曼不动杆菌的感染是一个不错的选择。     

Freeze-drying of Lactobacillus coryniformis Si3--effects of sucrose concentration, cell density, and freezing rate on cell survival and thermophysical properties

Freeze-drying is commonly used to stabilize lactic acid bacteria. Many factors have been reported to influence freeze-drying survival, including bacterial species, cell density, lyoprotectant, freezing rate, and other process parameters. Lactobacillus coryniformis Si3 has broad antifungal activity and a potential use as a food and feed biopreservative. This strain is considered more stress sensitive, with a low freeze-drying survival, compared to other commercialized antifungal lactic acid bacterial strains. We used a response surface methodology to evaluate the effects of varying sucrose concentration, cell density and freezing rate on Lb. coryniformis Si3 freeze-drying survival. The water activity of the dry product, as well as selected thermophysical properties of importance for freeze-drying; degree of water crystallization and the glass transition temperature of the maximally freeze concentrated amorphous phase (Tg') were determined. The survival of Lb. coryniformis Si3 varied from less than 6% to over 70% between the different conditions. All the factors studied influenced freeze-drying survival and the most important factor for survival is the freezing rate, with an optimum at 2.8 degrees C/min. We found a co-dependency between freezing rate and formulation ingredients, indicating a complex system and the need to use statistical tools to detect important interactions. The degree of water crystallization decreased and the final water activity increased as a function of sucrose concentration. The degree of water crystallization and Tg' was not affected by the addition of 10(8)-10(10) CFU/ml. At 10(11) CFU/ml, these thermophysical values decreased possibly due to increased amounts of cell-associated unfrozen water.     

Who's afraid of the big bad Wolff?: “Wolff's law” and bone functional adaptation

"Wolff's law" is a concept that has sometimes been misrepresented, and frequently misunderstood, in the anthropological literature. Although it was originally formulated in a strict mathematical sense that has since been discredited, the more general concept of "bone functional adaptation" to mechanical loading (a designation that should probably replace "Wolff's law") is supported by much experimental and observational data. Objections raised to earlier studies of bone functional adaptation have largely been addressed by more recent and better-controlled studies. While the bone morphological response to mechanical strains is reduced in adults relative to juveniles, claims that adult morphology reflects only juvenile loadings are greatly exaggerated. Similarly, while there are important genetic influences on bone development and on the nature of bone's response to mechanical loading, variations in loadings themselves are equally if not more important in determining variations in morphology, especially in comparisons between closely related individuals or species. The correspondence between bone strain patterns and bone structure is variable, depending on skeletal location and the general mechanical environment (e.g., distal vs. proximal limb elements, cursorial vs. noncursorial animals), so that mechanical/behavioral inferences based on structure alone should be limited to corresponding skeletal regions and animals with similar basic mechanical designs. Within such comparisons, traditional geometric parameters (such as second moments of area and section moduli) still give the best available estimates of in vivo mechanical competence. Thus, when employed with appropriate caution, these features may be used to reconstruct mechanical loadings and behavioral differences within and between past populations.     

Eimeria tenella: cloning of a novel Eimeria tenella cDNA encoding a protein related to rhomboid family from F2 hybrid strain

A novel cDNA sequence with an open reading frame of 774 bp from Eimeria tenella F2 hybrid strain (ETRH01) was isolated from a lambda cDNA library with a monoclonal antibody against sporozoite. Analysis of the genomic sequence suggests that this is an intronless gene. The deduced protein sequence has 257 amino acids with a calculated molecular weight of 28.349 kDa and an isoelectric point of 8.56. Sequence analysis revealed seven transmembrane domains and a rhomboid domain within the protein. RT-PCR result indicates that this gene was expressed in all of the five E. tenella isolates analyzed. To further study the role of this novel gene in the life cycle of E. tenella, ETRH01 was successfully expressed using pET28b(+) expression system.     

A phosphopantetheinyl transferase gene essential for biosynthesis of n-3 polyunsaturated fatty acids from Moritella marina strain MP-1

A phosphopantetheinyl transferase (PPTase) gene (pfaE), cloned from the docosahexaenoic acid (DHA)-producing bacterium Moritella marina strain MP-1, has an open reading frame of 861 bp encoding a 287-amino acid protein. When the pfaE gene was expressed with pfaA-D, which are four out of five essential genes for biosynthesis of eicosapentaenoic acid (EPA) derived from Shewanella pneumatophori SCRC-2738 in Escherichia coli, the recombinant produced 12% EPA of total fatty acids. This suggests that pfaE encodes a PPTase required for producing n-3 polyunsaturated fatty acids, which is probably involved in the synthesis of DHA in M. marina strain MP-1.     

A reappraisal of the role of abscisic acid and its interaction with auxin in apical dominance

BACKGROUND AND AIMS: Evidence from pea rms1, Arabidopsis max4 and petunia dad1 mutant studies suggest an unidentified carotenoid-derived/plastid-produced branching inhibitor which moves acropetally from the roots to the shoots and interacts with auxin in the control of apical dominance. Since the plant hormone, abscisic acid (ABA), known to inhibit some growth processes, is also carotenoid derived/plastid produced, and because there has been indirect evidence for its involvement with branching, a re-examination of the role of ABA in apical dominance is timely. Even though it has been determined that ABA probably is not the second messenger for auxin in apical dominance and is not the above-mentioned unidentified branching inhibitor, the similarity of their derivation suggests possible relationships and/or interactions. METHODS: The classic Thimann-Skoog auxin replacement test for apical dominance with auxin [0.5 % naphthalene acetic acid (NAA)] applied both apically and basally was combined in similar treatments with 1 % ABA in Ipomoea nil (Japanese Morning Glory), Solanum lycopersicum (Better Boy tomato) and Helianthus annuus (Mammoth Grey-striped Sunflower). KEY RESULTS: Auxin, apically applied to the cut stem surface of decapitated shoots, strongly restored apical dominance in all three species, whereas the similar treatment with ABA did not. However, when ABA was applied basally, i.e. below the lateral bud of interest, there was a significant moderate repression of its outgrowth in Ipomoea and Solanum. There was also some additive repression when apical auxin and basal ABA treatments were combined in Ipomoea. CONCLUSION: The finding that basally applied ABA is able partially to restore apical dominance via acropetal transport up the shoot suggests possible interactions between ABA, auxin and the unidentified carotenoid-derived branching inhibitor that justify further investigation.     

Application of a novel polyphasic approach to study the lactobacilli composition of sourdoughs from the Abruzzo region (central Italy)

AIMS: To characterize the lactobacilli community of 20 sourdoughs using a novel polyphasic approach. METHODS AND RESULTS: A polyphasic approach, consisting of a two-step multiplex polymerase chain reaction (PCR) system, 16S rRNA gene sequence analysis and physiological features, was applied to identify 127 isolates, representing about 37% of the presumptive lactobacilli collected from sourdough samples. Multiplex PCR successfully identified 111 isolates, while 16S rRNA gene sequencing was applied for the other 16 isolates, two of which could not be associated with any previously described lactic acid bacteria (LAB) species. Strain diversity was evaluated by phenotypic and random amplified polymorphic DNA-PCR analysis. Molecular detection of Lactobacillus group species was also performed on total DNA extracted from the doughs. CONCLUSIONS: Abruzzo region sourdough lactobacilli biodiversity, reflected in both Lactobacillus species composition and strain polymorphism, is similar to that of other Italian regions and is a source of novel LAB species. SIGNIFICANCE AND IMPACT OF THE STUDY: Within culture-independent methods, multiplex PCR is a rapid tool to study the lactobacilli population of sourdoughs.     

Inhibition of Listeria innocua and Brochothrix thermosphacta in vacuum-packaged meat by addition of bacteriocinogenic Lactobacillus curvatus CRL705 and its bacteriocins

AIMS: To evaluate the inhibition effectiveness of Lactobacillus curvatus CRL705 used as a bioprotective culture and of its bacteriocins, lactocin 705 and lactocin AL705, against Listeria innocua, Brochothrix thermosphacta and indigenous lactic acid bacteria (LAB) in vacuum-packaged meat stored at 2 degrees C. METHODS AND RESULTS: The live culture of Lact. curvatus CRL705 as well as synthetic lactocin 705 and purified lactocin AL705 were shown to be similarly effective in preventing the growth of B. thermosphacta and L. innocua in meat discs in contrast to control samples in which these micro-organisms grew rapidly, their numbers increasing by 3.0- and 2.1-log cycles respectively. In addition, indigenous LAB population showed a lower growth rate in the presence of lactocin 705. Bacteriocin activity was detected in the meat discs during 36 days at 2 degrees C irrespective of the biopreservation strategy applied. Changes in pH were not significantly different in meat discs treated with the protective culture when compared with control samples. CONCLUSIONS: Lactobacillus curvatus CRL705 and the produced bacteriocins, lactocin 705 and lactocin AL 705, were effective in inhibiting L. innocua and B. thermosphacta. The use of the bioprotective culture in refrigerated vacuum-packaged fresh meat would be more feasible from an economic and legal point of view. SIGNIFICANCE AND IMPACT OF THE STUDY: Establishment of biopreservation as a method to ensure the microbiological safety of vacuum-packaged fresh meat at 2 degrees C.