The continuous positive and negative dielectrophoresis of microorganisms

The continuous dielectrophoresis of living cells is described. The technique uses stream-centered transport of suspended microorganisms through an especially shaped non-uniform electric field. The cells can be given a positive or negative displacement, i.e., can be pushed into or out of the region of higher field intensity, depending upon the frequency of the applied ac field, and upon the relative permittivities of the cells and the suspending medium. Yeast (Saccharomyces cerevisiae) and algal cells (Chlorella vulgaris) were found to provide spectra of dielectrophoretic responses varying with the applied frequency (10 to 600 kHz) and conductivity.     

Cerium oxide and iron oxide nanoparticles abolish the antibacterial activity of ciprofloxacin against gram positive and gram negative biofilm bacteria

Metal oxide nanoparticles have been suggested as good candidates for the development of antibacterial agents. Cerium oxide (CeO₂) and iron oxide (Fe₂O₃) nanoparticles have been utilized in a number of biomedical applications. Here, the antibacterial activity of CeO₂ and Fe₂O₃ nanoparticles were evaluated on a panel of gram positive and gram negative bacteria in both the planktonic and biofilm cultures. Additionally, the effect of combining CeO₂ and Fe₂O₃ nanoparticles with the broad spectrum antibiotic ciprofloxacin on tested bacteria was investigated. Thus, minimum inhibitory concentrations (MICs) of CeO₂ and Fe₂O₃ nanoparticles that are required to inhibit bacterial planktonic growth and bacterial biofilm, were evaluated, and were compared to the MICs of the broad spectrum antibiotic ciprofloxacin alone or in the presence of CeO₂ and Fe₂O₃ nanoparticles. Results of this study show that both CeO₂ and Fe₂O₃ nanoparticles fail to inhibit bacterial growth and biofilm biomass for all the bacterial strains tested. Moreover, adding CeO₂ or Fe₂O₃ nanoparticles to the broad spectrum antibiotic ciprofloxacin almost abolished its antibacterial activity. Results of this study suggest that CeO₂ and Fe₂O₃ nanoparticles are not good candidates as antibacterial agents, and they could interfere with the activity of important antibiotics.     

革兰阴性菌引起的血培养阳性标本直接细菌鉴定和药敏的应用

目的 评估血培养阳性标本直接细菌鉴定和药敏可行性.方法 将血培养瓶放入Bact/Alert 3D 60血培养系统进行培养筛选.选取78份含革兰阴性杆菌的阳性血培养瓶进行试验.抽取培养液,用BD真空分离管离心血细胞.在收集到足量菌液后,用VITEK-32革兰阴性菌鉴定药敏卡做直接鉴定药敏试验.用标准方法及亚培养后的鉴定药敏试验对直接鉴定药敏试验进行评估.结果 VITEK-32直接鉴定试验,78株中的74株(94.9％)准确鉴定,直接药敏试验标准符合率95.6％.KB法血标本直接药敏试验标准符合率96.2％,但微小错误率高于VITEK-32直接药敏法.结论 Bact/Alert血培养阳性标本直接VITEK-32细菌鉴定和药敏对革兰阴性菌是切实可行的,可大幅度缩短时间,为临床及时修正用药提供依据,具有较高的应用价值.     

Higher order structure in the 3'-minor domain of small subunit ribosomal RNAs from a gram negative bacterium, a gram positive bacterium and a eukaryote

An experimental approach was used to determine and compare the highest order structure within the 150 to 200 nucleotides at the 3'-ends of the RNAs from the small ribosomal subunits of Escherichia coli, Bacillus stearothermophilus and Saccharomyces cerevisiae. Chemical reagents were employed to establish the degree of stacking and/or accessibility of each adenosine, guanosine and cytidine. The double helices were probed with a cobra venom ribonuclease from Naja naja oxiana, and the relatively unstructured and accessible sequences were localized with the single strand-specific ribonucleases A, T1, T2 and S1. The data enabled the various minimal secondary structural models, proposed for the 3'-regions of the E. coli and S. cerevisiae RNAs, to be critically examined, and to demonstrate that the main common features of these models are correct. The results also reveal the presence and position of additional higher order structure in the renatured free RNA. It can be concluded that a high level of conservation of higher order structure has occurred during the evolution of the gram negative and gram positive eubacteria and the eukaryote in both the double helical regions and the "unstructured" regions. Several unusual structural features were detected. Multiple G X A pairings in two of the putative helices, which are compatible with phylogenetic sequence comparisons, are strongly supported by the occurrence of cobra venom ribonuclease cuts adjacent to, and in one case between, these pairings. Evidence is also provided for the stacking of an A X A pair within a double helix of the yeast RNA. Other special structural features include adenosines bulged out from double helices; such nucleotides, which are hyper-reactive, have been implicated in protein recognition in 5 S ribosomal RNA. The 3'-terminal regions of the RNAs are particularly important for the functioning of the ribosome. They are involved in mRNA, tRNA and ribosomal factor binding. The results reveal that while the functionally important RNA sequences tend to be conserved, they are not always accessible in the free RNA; the pyrimidine-rich "Shine and Dalgarno" sequence, for example, which is involved in mRNA recognition, occurs in a double helix in both eubacterial RNAs.     

Extraction of crystal violet-iodine complex from gram positive bacteria by different solvents and its implication on gram differentiation

Summary Crystal violet from Gram stained S. aureus can be extracted completely by 95% ethanol if the stained bacteria is pre-treated with dilute sodium thiosulphate solution. Thiosulphate removes iodine form the cell component-dye-iodine complex instantaneously and renders the dye extractable by the differentiating medium. 11 alcoholic solutions of aniline, dimethyl aniline, nitro-benzene, benzene, toluene or xylol can also extract the color from the stained S. aureus; the extraction with the first three solvents is almost exhaustive while with the latter solvents extraction is appreciable but incomplete. These solvents can form charge-transfer complexes with iodine. The findings indicate that the stability of the cell component-dye-iodine complex determines the Gram-character of the cell. A model hasbeen presented for the Gram cell component-dye-iodine complex.     

Characterization of functional heme domains from soluble guanylate cyclase

Soluble guanylate cyclase (sGC) is a heterodimeric, nitric oxide (NO)-sensing hemoprotein composed of two subunits, alpha1 and beta1. NO binds to the heme cofactor in the beta1 subunit, forming a five-coordinate NO complex that activates the enzyme several hundred-fold. In this paper, the heme domain has been localized to the N-terminal 194 residues of the beta1 subunit. This fragment represents the smallest construct of the beta1 subunit that retains the ligand-binding characteristics of the native enzyme, namely, tight affinity for NO and no observable binding of O(2). A functional heme domain from the rat beta2 subunit has been localized to the first 217 amino acids beta2(1-217). These proteins are approximately 40% identical to the rat beta1 heme domain and form five-coordinate, low-spin NO complexes and six-coordinate, low-spin CO complexes. Similar to sGC, these constructs have a weak Fe-His stretch [208 and 207 cm(-)(1) for beta1(1-194) and beta2(1-217), respectively]. beta2(1-217) forms a CO complex that is very similar to sGC and has a high nu(CO) stretching frequency at 1994 cm(-)(1). The autoxidation rate of beta1(1-194) was 0.073/min, while the beta2(1-217) was substantially more stable in the ferrous form with an autoxidation rate of 0.003/min at 37 degrees C. This paper has identified and characterized the minimum functional ligand-binding heme domain derived from sGC, providing key details toward a comprehensive characterization.     

Characterization of particulate and soluble guanylate cyclases from rat lung.

Rat lung homogenates contained significant amounts of guanylate cyclase activity in both 100,000 times g (60 min) particulate and supernatant fractions. In the presence of detergent, the particulate fraction contained 40% as much activity as did the supernatant fraction. Detergent-dispersed particulate and partially purified soluble guanylate cyclase preparations were characterized with respect to divalent cation requirements, divalent cation interactions, kinetic behavior, and gel filtration profiles. Both soluble and particulate guanylate cyclases required divalent cation for activity. The soluble preparation was 10 times more active in the presence of Mn-2plus than in the presence of Mg-2plus or Ca-2plus and no detectable activity was seen with Ba-2plus or Sr-2plus. Particulate guanylate cyclase activity was detectable only in the presence of Mn-2plus. Both enzyme preparations required Mn-2plus in excess of GTP for optimal activity at subsaturating amounts of GTP. At near-saturating GTP, the soluble enzyme required excess Mn-2plus, but the particulate enzyme did not. For kinetic analyses the enzymes were considered to require two substrates: metal-GTP and Me-2plus. Apparent negative cooperative behavior was seen with the soluble enzyme when excess Mn-2plus (in excess of GTP) was varied from 0.01 to 0.2 mM; above 0.2 mM excess Mn-2plus classical kinetic behavior was seen with an apparent KMn-2plus of 0.2 mM at near-saturating MnGTP. Similar studies using the particulate preparation yielded only classical kinetic behavior, but the apparent KMn-2plus decreased to near zero when MnGTP was near-saturating. Kinetic patterns for the particulate and soluble enzymes also differed when reciprocal initial velocities were plotted as a function of reciprocal MnGTP concentrations; classical kinetic behavior was seen with the soluble enzyme with an apparent KMnGTP of about 12 muM (at near-saturating excess Mn-2plus), whereas apparent positive cooperative behavior was seen with the particulate preparation (Hill coefficient equals 1.6, S0.5 EQUALS 70 MUM. Ca-2plus activation of soluble guanylate cyclase was related to the Mn-2plus:GTP ratio. Activation was most apparent when saturating amounts of Mn-2plus and MnGTP. At relatively high concentrations of Ca-2plus (0.1 to 4 mM), the addition of 10 muM Mn-2plus resulted in a 3- to 5-fold increase in soluble guanylate cyclase activity. In contrast, Ca-2plus sharply inhibited particulate guanylate cyclase activity. Gel filtration profiles of particulate and soluble preparations indicated differences in physical properties of the enzymes. As estimated by gel filtration, particulate (detergent-dispersed)evels. Here, removal of renal tissue is contraindicated. In all renal hy     

Development of matrix lysis for concentration of gram positive bacteria from food and blood

The development of a fast, reliable and inexpensive protocol for the concentration of bacteria from food by the removal of fat, carbohydrates and proteins that is compatible with downstream alternative DNA-based quantification methods is described. The protocol was used for dairy products, cooked and smoked fish and meat, carbohydrate-rich cooked products, ready-to-eat sauces, egg and blood. Lysis resulted in pellets of reasonable size for further processing. Starch, plant materials, fungi, tissues such as sinew, and chalaza could not be dissolved. Using L. monocytogenes, S. aureus and B. cereus as model organisms, microscopic analysis of the remaining bacterial pellets revealed that the recovered bacteria remained physically intact, albeit that the viability of the cells was compromised. Using real-time PCR, 7.3 CFU of L. monocytogenes were detected in artificially contaminated ultra-high temperature treated (UHT) milk and raw milk.     

Utilization of the quantitative component of positive and negative results of short-term tests

In this paper we discuss the possibility of utilizing not only the qualitative component of the information obtained from long-term and short-term tests (as is customary), but also the quantitative component of the results. We suggest that there is probably a precise mathematical relationship between the qualitative and quantitative approaches. We show that utilizing the quantitative approach, it is possible to give confidence limits to a given prediction, a possibility potentially very useful for risk evaluation. We show that starting from a reasonable working hypothesis, it is possible to include even negative data in a unified quantitative approach. Incorporating the quantitative component of the information could offer appreciable gains in predictivity, especially when utilizing batteries of tests.     

Patterns of LPS synthesis in gram negative bacteria

Lipopolysaccharide (LPS), a lipid based carbohydrate polymer, is found in the outer membrane of Gram negative bacteria where it plays a vital role in its structure and function. It is chiefly responsible for the toxic effects of the bacterial diseases caused by these organisms and plays a role in the organisms defense against host immune attack. In recent experiments using high resolution gel techniques Goldman & Leive (1980) have revealed an unexpected heterogeneity in the distribution of polymer lengths found in the membrane with lengths ranging from 0 (lipid-A-core) to nearly 40 sugar units. Monomer units are an essential element of the synthesis process. Working with mutant strains of E. coli and Salmonella typhimurium, they have also shown that these bacteria will continue to synthesize LPS molecules with very long chains even though monomer unit production is severely reduced. The steps involved in the synthesis of LPS are known and in this paper it is shown that the results of Goldman & Leive cannot be obtained assuming the synthesis process is length independent as has been suggested. Moreover, the paradoxical persistence of long chains in spite of monomer suppression has a simple explanation once length dependence is assumed. These conclusions result from the analysis of a Markov chain model of synthesis.     

Nanoparticle-induced transition from positive to negative photochromism

We have designed and synthesized a photochromic spiropyran with a dithiolane appendage. The two sulfur atoms of the dithiolane ring encourage the adsorption of this compound on the surface of cadmium sulfide nanoparticles. The properties of the resulting photochrome-nanoparticle assemblies vary significantly with the experimental conditions selected for the preparation of the inorganic component. Nanoparticles prepared in the presence of tri-n-octylphosphine impose positive photochromism on the ligands. Instead, nanoparticles prepared in the presence of sodium dioctylsulfosuccinate impose negative photochromism on the ligand. This behavior is a consequence of the difference in the surface morphology of the two sets of nanoparticles. Indeed, emission spectra confirm the presence of surface defects on the nanoparticles exhibiting negative photochromism. Presumably, electrostatic interactions between these surface defects and the colored and zwitterionic isomer of the ligand are responsible for the transition from positive to negative photochromism. Thus, our studies demonstrate that the microscopic environment around a photochromic switch can regulate the relative stabilities of its colorless and colored states as well as their isomerization kinetics.     

Purification and properties of guanylate kinase from bovine retinas and rod outer segments

The presence of three soluble nucleotide phosphotransferases in bovine rod outer segments was demonstrated: guanylate kinase (EC 2.7.4.8), nucleoside-diphosphate kinase (EC 2.7.4.6) and adenylate kinase (EC 2.7.4.3). The enzyme guanylate kinase, which catalyzes the reaction GMP + ATP in equilibrium GDP + ADP, was purified to homogeneity from isolated bovine rod outer segments as well as from bovine retinas. The enzyme preparations obtained from both sources are identical in their chromatographic properties, molecular mass (20-23 kDa for both native enzyme and dodecylsulfate-denatured polypeptide), Km values (13 microM for GMP and 430 microM for ATP), specific activities, and nucleotide specificities. The enzyme's turnover number was estimated to be 130 s-1. The minimum amount of enzyme found in rod outer segments is about 1 copy per 800 rhodopsin molecules. The role of the enzyme in the cyclic GMP cycle in rod outer segments is discussed.