Flow cytometric investigation of filamentation, membrane patency, and membrane potential in Escherichia coli following ciprofloxacin exposure

Ninety-eight percent of the cells in a population of Escherichia coli in log-phase growth lost colony-forming ability after being exposed for 3 h to the quinolone antibiotic ciprofloxacin at four times the MIC in nutrient broth, a concentration easily reached in vivo. Flow cytometric analysis, however, demonstrated that only 68% of this bacterial population had lost membrane potential, as judged by the membrane potential-sensitive dye bis-(1,3-dibutylbarbituric acid) trimethine oxonol [DiBAC(4)(3)], and only 30% could no longer exclude the nucleic acid-binding dye propidium iodide (PI), reflecting lost membrane integrity, efflux mechanisms, or both. Subsequent removal of ciprofloxacin and resuspension in nutrient broth resulted in renewed cell division after 2 h, with a calculated postantibiotic effect (PAE) time of 57 min. The proportion of DiBAC- and PI-fluorescent cells in this recovering population remained stable for more than 4 h after antibiotic removal. Eighty percent of cells present at drug removal were filamentous. Their number subsequently decreased with time, and the increase in particle count seen at the end of the PAE resulted from the division of short cells. Exposure to ciprofloxacin in the presence of the protein synthesis inhibitor chloramphenicol increased colony-forming ability to 60% of starting population numbers. In contrast to ciprofloxacin alone, this antibiotic combination resulted in insignificant filamentation and no dye uptake. Subsequent drug removal and resuspension in nutrient broth resulted in the appearance of filaments within 1 h, with 69% of the population forming filaments at 3 h. Dye uptake was also seen, with 20% of the population fluorescing with either dye after 4 h. We were unable to relate dye uptake to the viable count. Cell division resumed 240 min after removal of both drugs, yielding a PAE calculated at 186 min. Inhibition of protein synthesis with chloramphenicol prevented ciprofloxacin-induced changes in bacterial morphology, cell membrane potential, and ability to exclude nucleic acid-binding dye. These changes persisted beyond the end of the classically defined PAE and were not a definite indicator of cell death as defined by loss of colony formation, which related at least in part to filamentation.     

A novel membrane protein, VanJ, conferring resistance to teicoplanin

Bacterial resistance to the glycopeptide antibiotic teicoplanin shows some important differences from the closely related compound vancomycin. They are currently poorly understood but may reflect significant differences in the mode of action of each antibiotic. Streptomyces coelicolor possesses a vanRSJKHAX gene cluster that when expressed confers resistance to both vancomycin and teicoplanin. The resistance to vancomycin is mediated by the enzymes encoded by vanKHAX, but not by vanJ. vanHAX effect a reprogramming of peptidoglycan biosynthesis, which is considered to be generic, conferring resistance to all glycopeptide antibiotics. Here, we show that vanKHAX are not in fact required for teicoplanin resistance in S. coelicolor, which instead is mediated solely by vanJ. vanJ is shown to encode a membrane protein oriented with its C-terminal active site exposed to the extracytoplasmic space. VanJ also confers resistance to the teicoplanin-like antibiotics ristocetin and A47934 and to a broad range of semisynthetic teicoplanin derivatives, but not generally to antibiotics or semisynthetic derivatives with vancomycin-like structures. vanJ homologues are found ubiquitously in streptomycetes and include staP from the Streptomyces toyocaensis A47934 biosynthetic gene cluster. While overexpression of staP also conferred resistance to teicoplanin, similar expression of other vanJ homologues (SCO2255, SCO7017, and SAV5946) did not. The vanJ and staP orthologues, therefore, appear to represent a subset of a larger protein family whose members have acquired specialist roles in antibiotic resistance. Future characterization of the divergent enzymatic activity within this new family will contribute to defining the molecular mechanisms important for teicoplanin activity and resistance.     

Multiparameter flow cytometric analysis of antibiotic effects on membrane potential, membrane permeability, and bacterial counts of Staphylococcus aureus and Micrococcus luteus

Although flow cytometry has been used to study antibiotic effects on bacterial membrane potential (MP) and membrane permeability, flow cytometric results are not always well correlated to changes in bacterial counts. Using new, precise techniques, we simultaneously measured MP, membrane permeability, and particle counts of antibiotic-treated and untreated Staphylococcus aureus and Micrococcus luteus cells. MP was calculated from the ratio of red and green fluorescence of diethyloxacarbocyanine [DiOC(2)(3)]. A normalized permeability parameter was calculated from the ratio of far red fluorescence of the nucleic acid dye TO-PRO-3 and green DiOC(2)(3) fluorescence. Bacterial counts were calculated by the addition of polystyrene beads to the sample at a known concentration. Amoxicillin increased permeability within 45 min. At concentrations of <1 microg/ml, some organisms showed increased permeability but normal MP; this population disappeared after 4 h, while bacterial counts increased. At amoxicillin concentrations above 1 microg/ml, MP decreased irreversibly and the particle counts did not increase. Tetracycline and erythromycin caused smaller, dose- and time-dependent decreases in MP. Tetracycline concentrations of <1 microg/ml did not change permeability, while a tetracycline concentration of 4 microg/ml permeabilized 50% of the bacteria; 4 microg of erythromycin per ml permeabilized 20% of the bacteria. Streptomycin decreased MP substantially, with no effect on permeability; chloramphenicol did not change either permeability or MP. Erythromycin pretreatment of bacteria prevented streptomycin and amoxicillin effects. Flow cytometry provides a sensitive means of monitoring the dynamic cellular events that occur in bacteria exposed to antibacterial agents; however, it is probably simplistic to expect that changes in a single cellular parameter will suffice to determine the sensitivities of all species to all drugs.     

Role of membrane immunoglobulin (Ig) crosslinking in membrane Ig-mediated, major histocompatibility-restricted T cell-B cell cooperation

Resting murine B lymphocytes can present rabbit anti-Ig to T cell lines specific for normal rabbit globulin. The T cell-B cell interaction is major histocompatibility complex (MHC)-restricted, and leads to activation, proliferation, and differentiation of the resting B cell into an antibody-secreting cell. Efficient antigen presentation and B cell activation depends upon binding of rabbit globulin to (membrane) mIg. To investigate the role of mIg in this polyclonal model for a T cell-dependent primary antibody response, we determined whether crosslinking of mIg is required either for efficient antigen presentation, as measured by helper T cell activation, or for the B cell response to T cell help, since all the direct effects of anti-Ig on B cells require crosslinking of mIg. We found that monovalent Fab' fragments of anti-IgM or anti-IgD work as efficiently as their divalent counterparts. Therefore, a signal transduced through the antigen receptor seems not to be required when T cell help is provided by an MHC-restricted T helper cell recognizing antigen on the B cell surface. Moreover, rabbit globulin bound to class I MHC molecules in the form of anti-H-2K also results in efficient antigen presentation and T cell-dependent B cell activation. However, mIg still appears to be specialized for antigen presentation, since anti-Ig is presented about three- to fivefold more efficiently than anti-H-2K.     

Novel membrane proteins present in teicoplanin-resistant, vancomycin-sensitive, coagulase-negative Staphylococcus spp.

Clinical isolates of Staphylococcus epidermidis and Staphylococcus haemolyticus resistant to teicoplanin (MIC 64 mg/L) and sensitive to vancomycin (MIC 2 mg/L), were compared with vancomycin- and teicoplanin-sensitive isolates (MICs 1 mg/L) of the same species. No apparent differences between the sensitive and resistant strains of either pair were found with respect to binding of teicoplanin to the bacteria, or to the amino acid content or degree of cross-linkage of purified peptidoglycan. The resistant strains did not inactivate teicoplanin in the surrounding medium. Analysis of the membrane proteins of the resistant S. epidermidis strain grown in the presence or absence of sub-inhibitory levels of teicoplanin (4 mg/L), showed the presence of a 39 kDa protein which was either absent, or present in considerably reduced amounts, in the sensitive strain. Fractionation of cell components after lysis of protoplasts showed that the 39 kDa protein was present predominantly in the membrane fraction but also in small amounts in the wall fraction. Similar investigations with S. haemolyticus revealed the presence of a 35 kDa protein in membranes of the resistant strain: the amount was increased substantially by growth in sub-inhibitory levels of teicoplanin. Membranes prepared by mechanical disintegration of bacteria or by osmotic lysis of protoplasts showed large apparent differences in the amounts of the 39 kDa protein.     

Autoantibodies, including antibasement membrane antibody, in patients with selective IgA deficiency

Laboratory data are presented on 48 patients with selective IgA deficiency. Sixty percent of the patients had autoimmune diseases or related disorders. We found antibasement membrane antibody (ABMA) in the sera of four IgA deficient patients and the incidence of ABMA seemed to be related to clinical symptoms. The incidence of various other antibodies was found to be increased in selective IgA deficiency. This phenomenon might be derived from the lack of local immune system. Inadequate IgA barrier might permit the exessive gastrointestinal absorption of food antigens or autoimmunogens in metabolic products which are excreted into the gut lumen.     

Identification of porins in outer membrane of Proteus, Morganella, and Providencia spp. and their role in outer membrane permeation of beta-lactams

Proteus mirabilis, Proteus vulgaris, Morganella morganii, Providencia rettgeri, and Providencia alcalifaciens, which were once classified into the same genus, Proteus, were studied. Cefoxitin-resistant mutants from these species were isolated, and it was confirmed that the resistance was attributed to the lack of an outer membrane protein, resulting in a significant decrease in the penetration of hydrophilic cephalosporins through the outer membrane. Comparison of the mutant strains with their parental strains in the diffusion rates of six monoanionic cephalosporins, a zwitterionic cephalosporin (cephaloridine), and a divalent anionic cephalosporin (cephalosporin C) suggested that each species had only one kind of porin protein, with molecular weights of 40,000 (Proteus mirabilis) or 37,000 (the other four species) and that the porins formed channels with cation selectivity, except for Proteus vulgaris. Porin proteins were purified from all the bacterial species except Providencia alcalifaciens, and the radius of the pores formed by the purified porins was estimated by the use of the liposome swelling assay. The pore radii were estimated to be approximately 0.59 nm (Proteus mirabilis), 0.63 nm (Proteus vulgaris), 0.58 nm (Providencia rettgeri), and 0.60 nm (M. morganii), similar to the size of the pore radius of Escherichia coli porins.     

The glomerular basement membrane: not gone, just forgotten

The glomerular capillaries function as the filtration barrier that retains albumin and other plasma proteins in the circulation. The unresolved question that has been asked for more than 50 years is, Which structural component of these capillaries constitutes the main molecular sieve that normally retains albumin and allows its passage in diseases associated with proteinuria? There is considerable evidence implicating both the glomerular basement membrane (GBM) and the epithelial filtration slits as the barrier. However, the prevailing point of view at present is that the slit diaphragms bridging the filtration slits are responsible for this important function, and evidence implicating the GBM is largely ignored or forgotten. In this issue of the JCI, Jarad et al. show that in laminin beta2-deficient (Lamb2-/-) mice, proteinuria can be directly attributed to the altered composition of the GBM (see the related article beginning on page 2272). Changes in the permeability of the GBM and its organization were primary to changes in the epithelium, as they preceded foot process effacement and loss of slit diaphragms.     

膜式氧合器中膜材料的研究进展

检索Pubmed数据库和万方数据库以及中国期刊全文数据库文献,分析膜式氧合器的特点.结果表明现在人工肺的应用主要是分离膜式,其形式已从最初的卷筒式、平板折叠式发展到今天广为采用的微孔中空纤维膜式.中空纤维膜氧合器使用中空纤维膜作为交换的主要场所,完成氧气和二氧化碳的交换,由于其在交换面积、氧合形态等方面具有优势,近年来已经成为人工肺研究的主要方向.用于制造中空纤维的材料主要为一些成纤性能良好的高分子材料,对膜材料的要求是具有良好的成膜性、热稳定性、化学稳定性、耐酸碱性、微生物侵蚀性和抗氧化性.当前的研究主要是为了提高气体交换能力和提高生物相容性,采用的方法主要有改进膜材料,优化设计以及对各种性能的实验评估和临床评价.     

Platelet membrane glycoproteins in thrombasthenia, Bernard-Soulier syndrome, and storage pool disease.

Quantitative polyacrylamide gel electrophoresis has been carried out on patients with Bernard-Soulier syndrome, Glanzmann's thrombasthenia, and storage pool defect in order to clarify the abnormalities in their platelet membrane glycoproteins. Normal individuals had values (expressed as PAS staining units/mg of membrane protein) of 5.11 +/- 0.63 for glycoprotein 1 (Mr 150,000), 2.35 +/- 0.35 for glycoprotein II (Mr 120,000), 0.89 +/- 0.22 for glycoprotein III (Mr 100,000), and 1.34 +/- 0.64 for glycoprotein IV (Mr 85,000). Total PAS staining of these four major bands was 9.70 +/- 1.26 PAS units/mg of membane protein. Patients with Bernard-Soulier syndrome completely lacked glycocalicin and had about one half (1.90 PAS units/mg) of the glycoprotein I of normal controls. These was no significant reduction in glycoproteins II, III, and IV, but total PASstaining was reduced to 4.40 units/mg, reflecting the importance of the contribution of glycoprotein I to this parameter. Thrombasthenic platelets gave values for glycoprotein II of 0.66, which were about 25% of controls, and the values for glycoprotein III (0.34) were about 40% of controls. Patients with storage pool disease gave values within the normal range with the exception of one family which showed, in addition, small platelets and an associated lipid defect. In thic case of glycoprotein (2.71) was significantly elevated.     

Glutathione peroxidase, thioredoxin, and membrane protein changes in erythrocytes predict ribavirin-induced anemia

OBJECTIVE: Low erythrocyte membrane protein sulfhydril concentrations are a risk factor for ribavirin-induced anemia. We further studied the role of oxidative stress and erythrocyte membrane alterations in ribavirin-induced anemia. METHODS: The levels of thioredoxin, glutathione peroxidase, protein sulfhydrils, and protein-mixed disulfides, as well as the electrophoretic membrane protein pattern, were determined in freshly isolated erythrocytes from healthy control subjects, patients without severe anemia during previous ribavirin treatment (still hepatitis C virus [HCV]-positive), and patients who had had severe anemia with ribavirin (still HCV-positive or HCV-negative), 6 months after full recovery. Erythrocytes were also incubated with buffer, ribavirin, phenylhydrazine, or dehydroepiandrosterone, and concentrations of protein sulfhydrils, protein-mixed disulfides, thiobarbituric acid-reactive substances, and total and oxidized glutathione, as well as osmotic resistance, were determined. RESULTS: Patients with previous severe ribavirin-induced anemia had lower levels of protein sulfhydrils (30.9 nmol/mg protein versus 43.2 nmol/mg protein, P<.001) and thioredoxin (0.6 nmol/g hemoglobin versus 1.2 nmol/g hemoglobin, P<.001), higher levels of protein-mixed disulfides (1.5 nmol/g hemoglobin versus 0.5 nmol/g hemoglobin, P<.001) and glutathione peroxidase (618 mU/mg protein versus 393 mU/mg protein, P<.001), and a membrane protein pattern consistent with band 4 dimer disaggregation. These differences were independent of HCV seropositivity. There were negative correlations between levels of glutathione peroxidase and thioredoxin (r=-0.87) and between levels of protein sulfhydrils and protein-mixed disulfides (r=-0.93). In vitro studies showed that erythrocytes of patients who had had hemolysis during treatment of HCV are more susceptible to oxidative stress. CONCLUSIONS: Pronounced differences in markers of oxidative stress and membrane proteins exist between patients with and without a history of ribavirin-induced anemia. Our findings suggest that there are erythrocyte-related risk factors for ribavirin-induced severe anemia.     

Functional reconstitution, membrane targeting, genomic structure, and chromosomal localization of a human urate transporter

Elevated serum levels of uric acid have been associated with an increased risk for gout, hypertension, cardiovascular disease, and renal failure. The molecular mechanisms for the diminished excretion of urate in these disorders, however, remain poorly understood. Human galectin 9, which is highly homologous to the rat urate transporter rUAT, has been reported to be a secreted or cytosolic protein. We provide data that galectin 9 is hUAT, the first identified human urate transporter. hUAT is a highly selective urate ion channel when inserted in lipid bilayers. When expressed in renal epithelial cells it is an integral plasma membrane protein with at least two transmembrane domains. The gene for hUAT consists of 11 exons and is mapped to chromosome 17; a highly homologous gene, hUAT2, maps to a nearby region of chromosome 17 and is also likely to be a urate transporter. hUAT is expressed in a wide variety of tissues and is present in at least three isoforms; hUAT2 is less widely expressed at severalfold lower levels than hUAT. Further knowledge about the functions of hUAT, its isoforms, and hUAT2, as well as mutational analysis of hUAT1 and hUAT2 in individuals or families with hyperuricemia, should significantly improve our understanding of the molecular mechanisms of urate homeostasis.     

New insights into the treatment of real N,N-dimethylacetamide contaminated wastewater using a membrane bioreactor and its membrane fouling implications

Treatment of N,N-dimethylacetamide (DMAC) wastewater is an important step in achieving the sustainable industrial application of DMAC as an organic solvent. This is the first time that treatment of a high concentration of DMAC in real wastewater has been assessed using membrane bioreactor technology. In this study, an anoxic–oxic membrane bioreactor (MBR) was operated over a month to mineralize concentrated DMAC wastewater. Severe membrane fouling occurred during the short-term operation of the MBR as the membrane flux decreased from 11.52 to 5.28 L (m² h)⁻¹. The membrane fouling was aggravated by the increased amount of protein fractions present in the MBR mixed liquor. Moreover, results from the excitation–emission matrix analysis identified tryptophan and other protein-like related substances as the major membrane-fouling components. Furthermore, analysis of the DMAC degradation mechanism via high performance liquid chromatography (HPLC) and ion chromatography (IC) revealed that the major degradation products were ammonium and dimethylamine (DMA). Although the MBR system achieved the steady removal of DMAC and chemical oxygen demand (COD) by up to 98% and 80%, respectively at DMAC₀ ≤ 7548 mg L⁻¹, DMA was found to have accumulated in the treated effluent. Our investigation provides insight into the prospect and challenges of using MBR systems for DMAC wastewater degradation.

Treatment of N,N-dimethylacetamide (DMAC) wastewater is an important step in achieving the sustainable industrial application of DMAC as an organic solvent.     

电子显微镜对实验动物消化器官、血管、神经、黏膜超微结构的观察

目的:通过对近10年来利用电子显微镜对实验动物消化器官、血管、神经、黏膜超微结构进行观察,探讨电子显微镜技术在动物消化器官结构中的应用。资料来源:手工检索吉林医药学院图书馆中,1995/2005与电子显微镜技术在动物消化器官结构中应用相关的文章,并限定语言种类为中文,检索词为“电子显微镜、电镜、消化器官、血管、神经、黏膜、超微结构”。资料选择:对资料进行初审,选取关于动物实验的文章,然后开始浏览全文,纳入内容为消化器官、血管、神经、黏膜的细胞超微结构、内壁的黏膜结构、微血管的超微结构、毛细淋巴管的超微结构、神经的超微结构以及一些病毒感染的细胞结构的文章,排除综述和重复性文章。资料提炼:共翻阅了9种相关杂志,收集到31篇符合要求的文章,其中与动物消化器官、血管、神经、黏膜的细胞超微结构相关的文章14篇,与毛细淋巴管的超微结构相关的文章5篇,与微血管的超微结构相关的文章4篇,与神经的超微结构相关的文章2篇,与内壁的黏膜结构相关的文章6篇。资料综合:随着电子显微镜分辨倍数、分辨率的提高,动物消化器官的研究也由简单的解剖学观察发展到超微结构的观察。在细胞的超微研究中看到了细胞的一些适于消化的功能,表明在动物从水到陆的进化进程中,消化道黏膜结构是由简单向复杂进化发展的。结论:这些利用超微结构研究出的成果为组织解剖学、生理学、病理学等方面的研究提供了重要依据和科学的方法,将为今后相关问题的研究提供形态学、组织解剖学、分类学等方面的依据。