Lysis of halophilic Vibrio alginolyticus and Vibrio costicolus induced by chaotropic anions.

High concentration (1.0 M) of KSCN, but not of NaSCN, induced lysis of slightly halophilic Vibrio alginolyticus and moderately halophilic Vibrio costicolus, and the decrease in absorbance of the cell suspension was complete after 30 min at 25 degrees C. Replacement of K+ with Na+ effectively prevented the lysis by SCN-.K+ salts of NO3-, Br- and I-, however, induced no significant lysis. In electron micrographs, a prolonged exposure of the cells of V. alginolyticus to 1.0 M KSCN displaced the nucleoplasm to maintain close contact with the cell membranes. After 40 min of interaction, 50% of the cellular protein, 96% of RNA and 94% of DNA were recovered in the lysed cells. In contrast to lysis in hypotonic conditions, the lysis induced by KSCN is due mainly to a partial release of protein from the cells. V. costicolus was more susceptible to SCN- than V. alginolyticus, whereas nonhalophilic Escherichia coli was resistant to 1.0 M KSCN. Thus, lysis by SCN- is characteristic of halophilic bacteria and cell membranes of more halophilic bacteria are more susceptible to chaotropic anions. The protective effect of Na+ observed here was considered to be manifested by specific interactions of Na+ with components of cell membranes, thereby rendering their structures resistant to the action of chaotropic anions.     

Isolation of Mycoplasma Membranes by Digitonin

The cell membrane of Mycoplasma hominis was isolated by lysing the cells with digitonin. Electron microscopy and chemical, density gradient, and electrophoretic analyses of the membrane proteins showed the membranes so obtained, like those isolated by osmotic lysis, to be relatively free of cytoplasmic contaminants. Sensitivity to digitonin lysis depended on temperature but was not affected by Mg(2+) ions and was only slightly affected by the age of the culture. Accordingly, it seems that digitonin may be used for the isolation of cell membranes from sterol-requiring mycoplasmas that tend to be fairly resistant to osmotic lysis.     

An ultrastructural study of HeLa cell invasion with Salmonella typhi GIFU 10007

Scanning electron micrograph of HeLa S3 monolayered cells, inoculated with viable bacteria of a Salmonella typhi strain GIFU 10007, revealed that the extended microvilli tangled the bacteria within 10 min after inoculation. The micrographs of HeLa cells, at 1 hr after inoculation, indicate the following: shortening of bacterium-attached microvilli, subsiding of tangled bacteria into microvilli bush, and then attachment of bacterial soma to cell surface making the cell membrane depressed. The transmission electron micrographs, at 1 hr after inoculation, demonstrated the findings of interaction between HeLa cell and S. typhi 10007, similar to those observed on scanning electron micrographs. Hair-like fine structures from the soma of challenge organisms were also observed. They were in contact with HeLa cell microvilli and cell membrane. The bacteria were first partially and then totally surrounded by the HeLa cell plasma membrane. One, two, or several bacteria with intact outer membrane were enclosed in intracytoplasmic membrane-bound vacuoles. Fragmented vacuolar membrane was still visible around the intracellularly accumulated bacteria at 24 hr after inoculation. The viable cells of S. typhi 10007 are regarded as internalizing into HeLa cells by a process of endocytosis and to multiply within the membrane-bound vacuoles.     

Three dimensional electrode array for cell lysis via electroporation

Microfabricated devices for cell lysis have demonstrated many advantages over conventional approaches. Among various design of microdevices that employ electroporation for cytolysis, most utilize Ag/AgCl wires or 2D planar electrodes. Although, simple in fabrication the electric field generated by 2D electrodes decays exponentially, resulting in rather non-uniform forcing on the cell membrane. This paper investigates the effect of electric field generated by 3D cylindrical electrodes to perform cell lysis via electroporation in a microfluidic platform, and compared with that by 2D design. Computational results of the electric field for both 2D and 3D electrode geometries showed that the 3D configuration demonstrated a significantly higher effective volume ratio-volume which electric field is sufficient for cell lysis to that of net throughflow volume. Hence, the efficacy of performing cell lysis is substantially greater for cells passing through 3D than 2D electrodes. Experimentally, simultaneous multi-pores were observed on leukocytes lysed with 3D electrodes, which is indicative of enhanced uniformity of the electric field generated by 3D design. Additionally, a single row of 3D electrode demonstrated a substantially higher lysing percentage (30%) than that of 2D (8%) under that same flow condition. This work should aid in the design of electrodes in performing cell lysis via electroporation.     

Evaluation of structural changes induced by high hydrostatic pressure in Leuconostoc mesenteroides

Scanning electron microcopy (SEM), transmission electron microscopy (TEM), and differential scanning calorimetry (DSC) were used to evaluate structural changes in Leuconostoc mesenteroides cells as a function of high-hydrostatic-pressure treatment. This bacterium usually grows in chains of cells, which were increasingly dechained at elevated pressures. High-pressure treatments at 250 and 500 MPa also caused changes in the external surface and internal structure of cells. Dechaining and blister formation on the surface of cells increased with pressure, as observed in SEM micrographs. TEM studies showed that cytoplasmic components of the cells were affected by high-pressure treatment. DSC studies of whole cells showed increasing denaturation of ribosomes with pressure, in keeping with dense compacted regions in the cytoplasm of pressure-treated cells observed in TEM micrographs. Apparent reduction of intact ribosomes observed in DSC thermograms was related to the reduction in number of viable cells. The results indicate that inactivation of L. mesenteroides cells is mainly due to ribosomal denaturation observed as a reduction of the corresponding peak in DSC thermograms and condensed interior regions of cytoplasm in TEM micrographs.     

Isolation and Characterization of Protoplasts from Saccharomyces rouxii

Cells of the osmotolerant yeast Saccharomyces rouxii were transformed to protoplasts in good yield (85%) by digesting cell walls with snail-gut enzyme in the presence of 10 mM dithioerythritol, 0.1 M sodium phosphate buffer (pH 6.8), and 2.0 M KCl. The requirement for 2.0 M KCl compares with that for S. bisporus var. mellis (another osmotolerant species) and contrasts with the 0.3 to 0.8 M KCl concentrations used in the preparation of most yeast protoplasts. Short digestions (60 min or less) produced mostly spheroplasts; longer incubations (90 min or more) yielded mostly protoplasts as judged by electron micrographs. These protoplasts could be transferred to 1.0 M KCl or 2.0 M sorbitol without lysing, but lysis was pronounced in 0.5 M KCl or 1.0 M mannitol and complete in 0.02 M KCl. Protoplasts were separated from isolated cell wall remnants and debris by centrifugation on a linear gradient of Ficoll 400 (35 to 17.5%, wt/vol) containing 2.0 M KCl. Both crude and fractionated protoplast preparations contained vesicles which were identified with the periplasmic bodies of whole cells. Some of the periplasmic bodies were connected to protoplasts by fine pedicels; others appeared free. Independent degeneracy of periplasmic bodies was occasionally observed. beta-Fructofuranosidase (EC 3.2.1.26) activity is cryptic (physically) in cells of S. rouxii in contrast to the expressed enzyme (periplasmic space) of other Saccharomyces species. This enzyme remains cryptic in protoplast preparations of S. rouxii but is expressed upon lysis. The same specific activities were found per unit cell or protoplast. The possible association of the cryptic enzyme with periplasmic bodies is discussed.     

Degradation of raw corn stover powder (RCSP) by an enriched microbial consortium and its community structure

A microbial consortium with a high cellulolytic activity was enriched to degrade raw corn stover powder (RCSP). This consortium degraded more than 51% of non-sterilized RCSP or 81% of non-sterilized filter paper within 8 days at 40 °C under facultative anoxic conditions. Cellulosome-like structures were observed in scanning electron micrographs (SEM) of RCSP degradation residue. The high cellulolytic activity was maintained during 40 subcultures in a medium containing cellulosic substrate. Small ribosomal gene sequence analyses showed the consortium contains uncultured and cultured bacteria with or without cellulolytic activities. Among these bacteria, some are anaerobic others aerobic. Analyses of the culture filtrate showed a typical anoxic polysaccharide fermentation during the culturing process. Reducing sugar concentration increased at early stage followed by various fermentation products that were consumed at the late stage.     

Evaluation of Structural Changes Induced by High Hydrostatic Pressure in Leuconostoc mesenteroides

Scanning electron microcopy (SEM), transmission electron microscopy (TEM), and differential scanning calorimetry (DSC) were used to evaluate structural changes in Leuconostoc mesenteroides cells as a function of high-hydrostatic-pressure treatment. This bacterium usually grows in chains of cells, which were increasingly dechained at elevated pressures. High-pressure treatments at 250 and 500 MPa also caused changes in the external surface and internal structure of cells. Dechaining and blister formation on the surface of cells increased with pressure, as observed in SEM micrographs. TEM studies showed that cytoplasmic components of the cells were affected by high-pressure treatment. DSC studies of whole cells showed increasing denaturation of ribosomes with pressure, in keeping with dense compacted regions in the cytoplasm of pressure-treated cells observed in TEM micrographs. Apparent reduction of intact ribosomes observed in DSC thermograms was related to the reduction in number of viable cells. The results indicate that inactivation of L. mesenteroides cells is mainly due to ribosomal denaturation observed as a reduction of the corresponding peak in DSC thermograms and condensed interior regions of cytoplasm in TEM micrographs.     

硫代磷酸反义寡脱氧核苷酸抗HBV细胞的实验研究

针对ＨＢＶ的５个基因位点作为靶序列,设计合成硫代反义寡聚核苷酸（Ｓ－ａｓＯＤＮ）．应用ＥＬＩＳＡ方法、ＭＴＴ比色法、电子显微镜等手段,观察Ｓ－ａｓＯＤＮ对ＨｅｐＧ２２２１５细胞ＨＢｓＡｇ、ＨＢｅＡｇ抗原的表达,及细胞的毒性、细胞形态和超微结构的影响．结果显示不同靶位点的Ｓ－ａｓＯＤＮ对ＨＢｓＡｇ、ＨＢｅＡｇ表达都有显著的抑制作用,且表现为序列特异性、剂量相关性、联合协同性和一定的抗核酸酶性,在浓度为２０μｍｏｌ／Ｌ时,对细胞无明显杀伤作用,对细胞的超微结构无显著的改变．结果提示Ｓ－ａｓＯＤＮ有望发展为抗ＨＢＶ的有效药物,但靶序列的选择、透细胞膜性及联合用药配伍等仍值得进一步研究和解决．     

Protection of cultured human monocytes from lymphokine-activated killer-mediated lysis by IFN-gamma

We have recently reported that IL 2-activated killer (LAK) cells are capable of lysing cultured human monocytes. In an effort to protect autologous monocytes from lysis, we treated monolayer cultures of adherent PBMC with various doses of human rIFN-gamma and assessed their susceptibility to LAK cells. IFN-gamma was shown to lessen the sensitivity of monocytes to lysis in a dose-dependent manner. Similar treatment of FMEX, an NK-resistant melanoma tumor cell line, with IFN-gamma did not affect its susceptibility to LAK lysis. Kinetic studies demonstrated that as little as 2 h incubation with IFN-gamma was sufficient for the protective effects to take effect. Additionally, monocytes that were pulsed with IFN-gamma for 2 h, washed, and then cultured in medium alone retained their resistance to lysis for at least 3 days. Cold target inhibition studies showed that IFN-treated and untreated monocytes could effectively compete with each other for binding sites on LAK cells. Furthermore, binding studies demonstrated that there was no significant difference between the number of conjugates formed by using either IFN-treated or untreated monocytes. This indicates that resistance to lysis induced by IFN treatment affects a post-binding event and not an initial recognition signal. From these studies, it was apparent that treatment of monocytes with IFN-gamma lessened their sensitivity to LAK-mediated lysis. Thus, it may be possible through a specific sequence of IFN-gamma and IL-2 treatment that LAK activity could be manipulated against some tumor cells, but not normal cells, to abrogate some of the toxicity seen with this type of cancer therapy.     

Generation of nonspecific murine cytotoxic T cells in vitro by purified human interleukin 2

Murine spleen cells developed into nonspecific cytotoxic cells within 72 hr of culture in the presence of highly purified sources of human interleukin 2. In whole spleen cell cultures, human interleukin 2 generated effector cells which were Thy 1.2⁺, Lyt 2.2⁺, resistant to γ irradiation (1000 R), and capable of lysing both H-2 compatible and incompatible targets. The effector cells generated in this manner were not restricted to classical natural killer cell-sensitive targets. If thymus-derived cells (T cells) were depleted from the spleen cell population before culture with human interleukin 2, the effector cells generated were enriched in effectors capable of lysing natural killer cell-sensitive targets. Interferon was not produced in interleukin 2-stimulated spleen cell cultures. In addition, heterologous antibody to murine -γ-interferon did not abrogate the generation of cytotoxic cells by human interleukin 2. These and additional data suggest that human interleukin 2 is capable of stimulating γ-irradiation-sensitive Thy 1.2⁺ cell(s) capable of lysing a variety of target cells regardless of inherent sensitivities to classical natural killer cells. Thy 1.2^? cells were also stimulated by human interleukin 2 and lysed only natural killer cell-sensitive targets. Human interleukin 2 caused some Thy 1.2^? cells to become susceptible to lysis by anti-Thy 1.2 serum and complement.     

抗CD3/抗CD20双特异性单链抗体介导的B淋巴瘤细胞体外裂解作用

在构建并成功表达抗CD3/抗CD20双特异性单链抗体(bscCD3×CD20)的基础上,对其在体外介导T淋巴细胞杀伤Ramous B淋巴瘤细胞的生物活性进行了分析。Annexin V/PI(AV/PI)染色和形态学观察及扫描电镜分析表明bscCD3×CD20介导的B淋巴瘤细胞体外裂解作用是通过先诱导靶细胞凋亡而继发坏死、裂解的方式实现的。非放射性细胞毒性分析表明bscCD3×CD20介导的T淋巴细胞杀伤活性随抗体浓度、反应时间和效靶比的升高而增加。在抗体浓度为5μg/mL、作用时间为24h、效靶比为10∶1时,杀伤活性最高可达87·3%。采用美国SuperArray人细胞凋亡芯片检测细胞杀伤起始阶段细胞凋亡相关基因的表达水平变化,许多凋亡相关基因的表达均发生了不同程度的上调或下调,其中ATM基因表达升高了187倍,p53基因升高了15倍,提示ATM-p53途径可能是bscCD3×CD20介导T细胞诱导B淋巴瘤细胞凋亡的主要途径。     

LYSIS OF MICROCYSTIS AERUGINOSA (KÜTZ.) BY BDELLOVIBRIO-LIKE BACTERIA1

Cells of Microcystis aeruginosa (Kützing), collected from water-blooms of Lake Varese, were lysed by Bdellovibrio-like bacteria. The cells were lysed only after penetration. The cyanobacteria and lysing bacteria were characterized by a fibrous glycocalyx. Once the host cell was penetrated, the bacteria remained localized mainly between the host cell wall and cytoplasmic membrane, which appeared partially thickened. Cell lysis began by breakdown of cell structures. The cell wall appeared broken at many sites, and in completely lysed cells, was partially interrupted. The lysis of Microcystis by bacteria could be one of the causes of the death of algal blooms.     

Factors determining the ability of cytokine-activated killer cells to lyse human ovarian carcinoma targets

Lysis of human ovarian carcinoma cells by natural killer (NK) cells, interferon-alpha activated NK cells (alpha-NK) and lymphokine-activated killers cells (LAK) was studied using both fresh tumor cells and a cell line (HEY) as targets. A clonogenic assay to measure cell kill was more sensitive than a 4-h 51Cr release assay. Both assays showed that single cells were more effectively lysed than were tumor clumps (spheroids). Freshly isolated tumor cells studied in the 51Cr release assay appeared equally susceptible to lysis by LAK cells whether in the form of clumps or single cells, but NK and alpha-NK effectors appeared much less effective in lysing susceptible target cells when they were in clumps. Tumor cells from some patients showed marked resistance to lysis by NK and alpha-NK cells in fractions enriched for clonogenic cells, even when tested in a single cell-suspension, whereas LAK cells were always cytolytic. These data suggest that intrinsic resistance of ovarian carcinoma to lysis by LAKs is unlikely to explain failure of LAK + IL-2 therapy to eradicate tumor in vivo.     

Gene mapping and somatic cell hybrid analysis of the role of human lymphocyte function-associated antigen-3 (LFA-3) in CTL-target cell interactions

LFA-3 is expressed on a wide variety of human cell lines, including those which have been used as recipients for gene transfer of human class I gene products, whereas a murine counterpart is either absent or significantly different such that the anti-LFA-3 monoclonal antibody (MAb) does not bind. By using a somatic cell genetic approach, we demonstrate that LFA-3 is not a major histocompatibility complex-encoded molecule, and that its gene locus maps to human chromosome 1. When LFA-3 and HLA-A2 are coexpressed on the mouse cell surface, anti-LFA-3 MAb interfered with specific recognition and lysis of these target cells by human CTL capable of lysing HLA-A2-expressing mouse transfectants. A significant contribution of the LFA-3 molecule to CTL reactivity was not observed, however, because the presence of LFA-3 did not restore recognition by CTL clones previously found incapable of lysing HLA-A2-expressing mouse transfectants, nor was it required by those human CTL that could lyse mouse cell transfectants. Thus, we have used genetic techniques to demonstrate that LFA-3 may serve a role in CTL-target cell interactions at the target cell level, but is not a molecule absolutely required for human allospecific CTL recognition of HLA antigens expressed on mouse cells. We suggest that LFA-3 may not participate directly in CTL function under normal circumstances, but delivers a more general inhibitory signal only when provoked by bound MAb.     

Correlation of protein electrophoretic pattern with morphology of normal and mutant feathers

Differences are demonstrated in electrophoretic patterns of SCM proteins extracted from the shaft and vane between the plumulaceous and pennaceous portions of normal feathers. Supportive evidence for these differences is given by scanning electron micrographs. In various mutant feathers, the observed structural and electrophoretic differences were due to the distribution of plumulaceous and pennaceous parts, not to new proteins. Feather mutants appear to be due to regulatory gene changes rather than to structural gene products.Supported by NSF Grant BO-20086.     

Tumour-infiltrating lymphocytes mediate lysis of autologous squamous cell carcinomas of the head and neck

Tumour-infiltrating lymphocytes (TIL) and tumours from six patients with squamous cell carcinomas of the head and neck (SCCHN) were investigated. The six tumours all expressed major histocompatibility complex (MHC) class I antigens both in vivo and as tumor cell lines grown in vitro. In addition, the cancer cells either overexpressed the tumour-suppressor gene product p53 or harboured human papilloma virus 16/18 (HPV). The TIL were expanded in vitro in the presence of interleukin-2, immobilised anti-CD3 mAb and soluble anti-CD28 mAb. Expanded TIL cultures contained both CD4+and CD8+T cells, but generally contained few CD56+CD3-cells of the natural killer (NK) phenotype. CD8+T cells dominated the individual TIL cultures from five of the six patients and showed significant autologous tumour cell lysis. In TIL cultures derived from four of these tumour-reactive TIL cultures, killing could be partially blocked by an anti-MHC class I mAb. TIL cultures reacting with autologous tumour cells also showed strong TCR/CD3-redirected cytotoxicity when assayed against hybridoma cells expressing anti-TCR/CD3 mAb as well as natural-killer(NK)-like activity. A number of TIL cultures devoid of autologous tumour cell lysis were capable of lysing the natural-killer(NK)-sensitive K562 cell line suggesting that the SCCHN cells themselves are resistant to NK-like lysis. In conclusion, TIL cultures from head and neck carcinomas contain T cells which, upon expansion in vitro, can lyse autologous tumour cells in a MHC-class-I-restricted fashion. Thus, the results of the present study document that carcinomas of the head and neck in some patients are infiltrated by cytotoxic T cell precursors potentially capable of rejecting the autologous tumour.     

Alkaline lysis of mammalian cells for sedimentation analysis of nuclear DNA. Conformation of released DNA as monitored by physical, electron microscopic and enzymological techniques.

The degree of single strandedness of the DNA released from rat liver nuclei by various alkaline lysing solutions (including some with sodium dodecyl sulfate) was determined both before and after sedimentation in alkaline sucrose gradients employing electron microscopy, melting profiles, circular dichroism measurements, and digestibility by S1 nuclease. Regardless of the technique employed, the results obtained following alkaline sucrose gradient centrifugation of the DNA are consistent. The DNA was completely single stranded as judged by electron microscopy, circular dichroism spectra, and digestibility by S1 nuclease, an enzyme that specifically hydrolyzes single-stranded DNA. This was not true if the DNA was analyzed following alkaline lysis of the nuclei but before centrifugation. Under conditions which gave a complete transition to the single-stranded state, as judged by melting profiles and circular dichroism spectra, only 10-15% of the DNA was hydrolyzed by S1 nuclease. An increase in the susceptibility of the released DNA to S1 nuclease was observed with increases in the pH of the lysing solution. In order to release DNA which was single stranded as judged by both physical and enzymological techniques, the rat liver nuclei were lysed for 30 min with a 0.3 M NaOH lysing solution containing 0.5% dodecyl sulfate, 0.3 M NaCl and 0.03 M EDTA.     

Ultraviolet-B Induced Changes in Ultrastructure and D1/D2 Proteins in Cyanobacteria Synechococcus sp. PCC 7942

Effects of ultraviolet-B (UV-B) irradiation on ultrastructure, total cellular protein, and PS2 proteins D1 and D2 of Synechococcus sp. PCC 7942 cells was studied. The scanning electron micrographs showed UV-B radiation induced bending of the cells. The transmission electron micrographs revealed disorganization and shift in thylakoid lamellar structure to one side of the cell. The cellular phycocyanin/chlorophyll ratio decreased with increasing UV-B treatment and due to this the colour of cells turned light-green. No apparent change in total cellular proteins was evident, but the contents of two major proteins of PS2, D1 and D2, showed decline due to UV-B irradiation, although to different extent.     

Confocal scanning light microscopy of the Escherichia coli nucleoid: comparison with phase-contrast and electron microscope images.

The nucleoid of living and OsO4- or glutaraldehyde-fixed cells of Escherichia coli strains was studied with a phase-contrast microscope, a confocal scanning light microscope, and an electron microscope. The trustworthiness of the images obtained with the confocal scanning light microscope was investigated by comparison with phase-contrast micrographs and reconstructions based on serially sectioned material of DNA-containing and DNA-less cells. This comparison showed higher resolution of the confocal scanning light microscope as compared with the phase-contrast microscope, and agreement with results obtained with the electron microscope. The effects of fixation on the structure of the nucleoid were studied in E. coli B/r H266. Confocal scanning light micrographs and electron microscopic reconstructions showed that the shape of the nucleoid remained similar after OsO4 or glutaraldehyde fixation; however, the OsO4 nucleoid appeared to be somewhat smaller and more centralized within the cell.