The antagonistic activity of bifidobacteria in vitro and in vivo studied by using gnotobiological technology

The antagonistic activity of 4 strains of bifidobacteria (B. adolescentis 2 F1, B. longum Z4, B. breve R2 and B. bifidum G1), isolated from the vagina of healthy females of the reproductive age, with respect to Escherichia coli, Klebsiella ozaenae, Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa and Gardnerella vaginalis were studied in vitro and in vivo. The in vitro experiments revealed that all above-mentioned bifidobacteria were capable of inhibiting the growth of all indicator bacterial strains. Still of all the bifidobacteria under study had different levels of activity. B. adolescentis strain 2 F1 exhibited the highest inhibiting activity in vitro. In contrast to in vitro experiments, in vivo experiments with B. breve R2 demonstrated its high antagonistic activity with respect to E. coli. The data thus obtained indicate that in the study of antagonistic activity the use of the in vivo model as also expedient, for it is mainly in vivo that probiotic preparations show their activity.     

Genotoxicity testing of fluconazole in vivo and in vitro

The genotoxic effects of the antifungal drug fluconazole (trade name triflucan) were assessed in the chromosome aberration (CA) test in mouse bone-marrow cells in vivo and in the chromosome aberration, sister chromatid exchange (SCE) and micronucleus (MN) tests in human lymphocytes. Fluconazole was used at concentrations of 12.5, 25.0 and 50.0 mg/kg for the in vivo assay and 12.5, 25.0 and 50.0 microg/ml were used for the in vitro assay. In both test systems, a negative and a positive control (MMC) were also included. Six types of structural aberration were observed: chromatid and chromosome breaks, sister chromatid union, chromatid exchange, fragments and dicentric chromosomes. Polyploidy was observed in both the in vivo and in vitro systems. In the in vivo test, fluconazole did not significantly increase the frequency of CA. In the in vitro assays, CA, SCE and MN frequencies were significantly increased in a dose-dependent manner compared with the negative control. The mitotic, replication and cytokinesis-block proliferation indices (CBPI) were not affected by treatments with fluconazole. According to these results, fluconazole is clastogenic and aneugenic in human lymphocytes, but these effects could not be observed in mice. Further studies should be conducted in other test systems to evaluate the full genotoxic potential of fluconazole.     

双歧杆菌体外对Caco-2的黏附及其表面性质分析

【目的】体外测定双歧杆菌的黏附能力并对其表面性质进行分析。【方法】利用Caco-2细胞作为黏附模型体外测定七株菌的黏附能力,同时分析其自动聚集能力和表面疏水性,通过采用不同酶及化学物质处理双歧杆菌菌体细胞表面初步确定双歧杆菌细胞表面黏附相关化合物的类型,并对双歧杆菌表面蛋白进行电泳分析。【结果】自动聚集能力和表面疏水性均高的双歧杆菌菌株,其黏附能力高于自动聚集能力和表面疏水性均低的菌株,表现出明显的正相关。此外,受试菌株的黏附能力对蛋白酶和高碘酸钠敏感,利用LiCl对菌体表面蛋白进行提取后,其黏附能力明显下降,SDS-PAGE结果表明LiCl提取物中含有分子量大小不等的多个蛋白。【结论】双歧杆菌体外对Caco-2细胞的黏附具有菌株特异性,其黏附能力与表面疏水性质和自动聚集能力相关,此外,推测双歧杆菌表面可能含有能调节其黏附的糖蛋白类物质。     

Lamellar lubrication in vivo and vitro: friction testing of hexagonal boron nitride

Phospholipid molecules (PLs) in vivo and graphite, molybdenum disulfide, tungsten disulfide and hexagonal boron nitride (h-BN) in vitro are good examples of frictionless lubricants. PLs and solid materials have the ability to form multi-bilayer or layered structures similar to lamellate solid. It has been confirmed experimentally that PLs as lamellar lubricants protect the surface of joints against wear while acting as frictionless lubricant. An experimental study has been conducted on the friction properties of h-boron nitride on porous non-full journal bearings. The porous non-full journal bearings were a mixture of 97.5 wt.% Fe and 2.5 wt.% Cu powder, and compressed to a density of 5.9 g/cm(3). The porosity of non-full journal bearings were 15.5 and 27.8 wt.% and were impregnated with vaseline and vaseline+5 wt.% h-BN. Additionally, the two additives SFR NLGI #2 (or SFR 2522) grease and graphite grease were used for comparison to h-BN. The tribological tests were performed on a four-ball machine under load of 49 daN, and a friction tribotester. The above experiment strongly suggested that h-BN has the ability to lubricate under load with very low friction coefficient comparable to phospholipids. Relatively low surface energy and low adhesion between the crystallites are giving the additives low friction coefficient. The results of the experimental studies showed that h-BN as an additive in vaseline possesses friction reducing properties, and excellent anti-wear properties.     

Lamellar lubrication in vivo and vitro: Friction testing of hexagonal boron nitride

Phospholipid molecules (PLs) in vivo and graphite, molybdenum disulfide, tungsten disulfide and hexagonal boron nitride (h-BN) in vitro are good examples of frictionless lubricants. PLs and solid materials have the ability to form multi-bilayer or layered structures similar to lamellate solid. It has been confirmed experimentally that PLs as lamellar lubricants protect the surface of joints against wear while acting as frictionless lubricant. An experimental study has been conducted on the friction properties of h-boron nitride on porous non-full journal bearings. The porous non-full journal bearings were a mixture of 97.5 wt.% Fe and 2.5 wt.% Cu powder, and compressed to a density of 5.9 g/cm³. The porosity of non-full journal bearings were15.5 and 27.8 wt.% and were impregnated with vaseline and vaseline + 5 wt.% h-BN. Additionally, the two additives SFR NLGI #2 (or SFR 2522) grease and graphite grease were used for comparison to h-BN. The tribological tests were performed on a four-ball machine under load of 49 daN, and a friction tribotester. The above experiment strongly suggested that h-BN has the ability to lubricate under load with very low friction coefficient comparable to phospholipids. Relatively low surface energy and low adhesion between the crystallites are giving the additives low friction coefficient. The results of the experimental studies showed that h-BN as an additive in vaseline possesses friction reducing properties, and excellent anti-wear properties.     

Bridging the gap between in vitro and in vivo toxicology testing

In vitro systems for neurotoxicological studies can be useful for the investigation of events associated with pertubations of cellular and molecular targets that are similar to those in the intact animal. The toxicities of organophosphorus compounds, which inhibit esterases, and 1,2,3,6-tetrahydropyridine (MPTP), which depletes dopamine, can be studied in human neuroblastoma cells.     

Fifty years of brain tissue mechanical testing: from in vitro to in vivo investigations

Beginning in the 1960s many studies have been performed to investigate the mechanical properties of brain. In this paper we point out the difficulties linked with in vitro experimental protocols as well as the advantages of using recently developed non-invasive in vivo techniques, such as magnetic resonance elastography. Results of in vitro and in vivo work are compared, emphasizing the specificities and disparities of the in vitro as well as the in vivo results. In particular, a detailed discussion of the results obtained from dynamic shear experiments and magnetic resonance elastography is given before arriving at a tentative conclusion on the state of knowledge of the mechanical properties of brain.     

Methods and goals for the use of in vitro and in vivo chemosensitivity testing

Sensitive, specific, and accurate methods to assay chemosensitivity are needed to (1) screen new therapeutic agents, (2) identify patterns of chemosensitivity for different tumor types, (3) establish patterns of cross-resistance and sensitivity in treatment of naïve and relapsing tumors, (4) identify genomic and proteomic profiles associated with sensivity, (5) correlate in vitro response with preclinical in vivo effects and clinical outcomes for a particular therapeutic agent, and (6) tailor chemotherapy regimens to individual patients. Various methods are available to achieve these end points, including several in vitro clonogenic and proliferation assays, cell metabolic activity assays, molecular assay to monitor expression of markers for responsiveness, drug resistance, and for induction of apoptosis, in vivo tumor growth and survival assays in metastatic and orthotopic models, and in vivo imaging assays. The advantages and disadvantages of the specific assays are discussed. A summary of research questions related to chemosensitivity testing is also included.     

New selective medium for selection of bifidobacteria from human feces

This work reports an alternative selective medium for reliable and efficient isolation of human fecal bifidobacteria. It uses a base commercially available, does not need pH adjustment and can be autoclaved with its additives. It provides a useful alternative for fecal bifidobacteria isolation.     

Correlation between in vitro mucus adhesion and the in vivo colonization ability of lactic acid bacteria: screening of new candidate carp probiotics

We measured the adhesion of candidate probiotic lactic acid bacteria (LAB) to carp intestinal mucus. The percentage of adherent bacteria varied among strains. Four strains, two with high adhesion and two with low adhesion in vitro, were tested for in vivo colonization ability. Carp were fed LAB-containing feed for 12 d, and then unsupplemented feed until day 33, and the numbers and compositions of intestinal LAB were analyzed during the entire period. LAB with lower in vitro adhesion disappeared quickly from the intestine after LAB feeding stopped. LAB with higher in vitro adhesion remained in the intestine 3 weeks after LAB feeding stopped, indicating a strong correlation between mucus adhesion in vitro and colonization ability in vivo. Next we isolated nine candidate probiotic LAB with high in vitro mucus-binding ability. Three of them were fed to carp, and all three were stably maintained in the intestine.     

Minimizing errors during in vitro testing of multisegmental spine specimens: considerations for component selection and kinematic measurement

Apparatus-induced artifacts may invalidate standard spine testing protocols. Kinematic measurements may be compromised by the configuration of motion capture equipment. This study has determined: (1) the influence of machine design (component friction) on in vitro spinal kinetics; (2) the sensitivity of kinematic measurements to variations in the placement of motion capture markers. A spinal loading simulator has been developed to dynamically apply pure bending moments (three axes) with or without a simultaneous compressive preload. Two linear slider types with different friction coefficients, one with caged ball bearings and one with high-precision roller bearings on rails, were mounted and specimen response compared in sequential tests. Three different optoelectronic marker cluster configurations were mounted on the specimen and motion data was captured simultaneously from all clusters during testing. A polymer tube with a uniform bending stiffness approximately equivalent to a polysegmental lumbar spine specimen was selected to allow reproducible behavior over multiple tests. The selection of sliders for linear degrees of freedom had a marked influence on parasitic shear forces. Higher shear forces were recorded with the caged-bearing design than with the high-precision rollers and consequently a higher moment was required to achieve a given rotation. Kinematic accuracy varied with each marker configuration, but in general higher accuracy was achieved with larger marker spacings and situations where markers moved predominantly parallel to the camera's imaging plane. Relatively common alternatives in the mechanical components used in an apparatus for in vitro spine testing can have a significant influence on the measured kinematic and kinetics. Low-magnitude parasitic shear forces due to friction in sliders induces a linearly increasing moment along the length of the specimen, precluding the ideal of pure moment application. This effect is compounded in polysegmental specimens. Kinematic measurements are highly sensitive to marker design and placement, despite equivalent absolute precision of individual marker measurements, however marker configurations can be designed to minimize errors related to spatial distribution and system bias.     

In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo

One approach to targeted therapies for cardiovascular disease relies on isolating ligands that enhance the tissue-specific uptake of genes or drugs by heart cells. To obtain heart-targeting ligands, phage display biopanning was used to isolate a 20-mer peptide that binds to isolated primary cardiomyocytes. The isolated phage, PCM.1, displays the peptide WLSEAGPVVTVRALRGTGSW, and binds these cells 180 times better than a control phage from the library. Furthermore, phage displaying this peptide preferentially bind to cardiomyocytes when compared with a panel of other cell types. A BLAST search revealed that this peptide contains a 12 amino acid segment with sequence identity to a peptide in tenascin-X, an extracellular matrix protein. Synthetic peptides containing the complete 20-mer or a 12-mer tenascin peptide partially blocked phage binding to the cardiomyocytes. We developed a quantitative real-time PCR assay to assess uptake of this phage by tissues in vivo. Using this assay, preferential localization of the PCM.1 phage in heart was observed compared to the uptake of this phage by other tissues or other phage by heart. Furthermore, PCM.1 phage was associated with cardiomyocytes isolated from mice treated with a phage in vivo. These results demonstrate the utility of biopanning on isolated cells for identifying specific binding peptides that can target a tissue in vivo.     

Novel amidino substituted 2-phenylbenzothiazoles: Synthesis,antitumor evaluation in vitro and acute toxicity testing in vivo

The efficient synthesis of new bis-substituted nitro-amidino, amino-amidino (10a, 10b–13a, 13b) and previously prepared diamidino 2-phenyl-benzothiazoles (9a, 9b) is described. The compounds 11a and 11b were prepared by recently developed methodology of the key precursors in zwitterionic form 8a and 8b with 4-nitrobenzoylchloride in a very good yield (70%). All compounds except diamidino-substituted 2-phenylbenzothiazole 9a show exceptionally prominent tumor cell-growth inhibitory activity and cytotoxicity, whereby the special selectivity of amino-amidine 2-phenylbenzothiazole 12a towards MCF-7 and H 460 cells makes this compound a prospective lead compound that should be further evaluated in animal models. All in vivo tested compounds (12a, 12b, 13a and 13b) are absorbed from mice gastrointestinal system. LD₅₀ are between 67.33 and 696.2 mg/kg body weight (OECD/EPA toxicity categories 2–3).     

Determinants of Escherichia coli RNase P cleavage site selection: a detailed in vitro and in vivo analysis.

The location of the Escherichia coli RNase P cleavage site was studied both in vitro and in vivo. We show that selection of the cleavage site is dependent on the nucleotide at the cleavage site and the length of the acceptor-stem. Within the acceptor-stem the number of nucleotides on the 5'-half of the acceptor-stem appears to be the important determinant, rather than the number of base pairs in the acceptor-stem. We also demonstrate that the length of the T-stem and a G to C substitution at position 57 in the tRNA(Tyr)Su3 precursor influence the location of the cleavage site under certain conditions. With respect to the function of the subunits of RNase P our data suggest that the nucleotide at position 333 in M1 RNA, and the C5 protein, are important for the identification of the cleavage site.     

体外去除胆固醇菌株的筛选及其作用机理研究

从青年人肠道中筛选分离,鉴定得到8株乳杆菌,进行胆固醇去除、耐酸和耐胆汁盐实验,发现两株植物乳杆菌Lp529和Lp501同时具有较高的体外去除胆固醇能力和耐胆汁盐及耐酸性能。通过对它们胆固醇去除过程和胆固醇在相应固液相中分布情况的实验分析,表明：Lp501与Lp529去除胆固醇的机理有差别,存在菌株特异性。研究还发现,被菌株吸收的胆同醇没有被代谢为其他物质,并可在条件适合时重新释放出来。     

In vivo versus in vitro screening or selection for catalytic activity in enzymes and abzymes

The recent development of catalytic antibodies and the introduction of new techniques to generate huge libraries of random mutants of existing enzymes have created the need for powerful tools for finding in large populations of cells those producing the catalytically most active proteins. Several approaches have been developed and used to reach this goal. The screening techniques aim at easily detecting the clones producing active enzymes or abzymes; the selection techniques are designed to extract these clones from mixtures. These techniques have been applied both in vivo and in vitro. This review describes the advantages and limitations of the various methods in terms of ease of use, sensitivity, and convenience for handling large libraries. Examples are analyzed and tentative rules proposed. These techniques prove to be quite powerful to study the relationship between structure and function and to alter the properties of enzymes.     

Cell selection in vivo

Summary A cytogenetic follow-up has been made of nine mixoploid children found among 11 148 consecutive newborn children.The frequency of the cell line with normal chromosomes increased in all but two, and the increase was statistically significant, being from 20% to 39% in four cases, and from 1% to 17% in three, while in one case there was no difference from the first to the last examination. The possibility that children with mixoploid chromosome abnormalities at birth will reveal no cell line with a chromosome abnormality in lymphocyte cultures as adults, despite having clinical signs of the chromosome aberration found in one cell line at birth is discussed, as is the question of cell selection in vivo.The mixoploid children had fewer clinical symptoms and fewer signs of the chromosome abnormalities found in some of their cells than children with the same chromosome abnormalities in all cells.