Laser tweezers Raman spectroscopy potential for studies of complex dynamic cellular processes: single cell bacterial lysis.

The potential of laser tweezers Raman spectroscopy (LTRS) to study complex and dynamic cellular processes was investigated on the model of single E. coli cells lysed (1) from "outside" with egg white lysozyme and (2) from "within" by temperature-induced temperate bacteriophage lambdacI857. The two lysis processes differed in the final outcome (incomplete vs complete cell lysis) as revealed by the dynamic laser light scattering and exhibited distinctive dynamic Raman spectra changes. The technique enabled for the first time at the cellular level to observe and quantify real time interaction of lysozyme with E. coli cells, "visualize" a side effect of the process due to the presence of EDTA, and correlate the process of cell wall disruption, as evidenced by the onset and development of asymmetric speckle scattering patterns, with release/escape of intracellular material (ribosomes, nucleic acids, proteins, etc.) quantified by the intensity changes of Raman signatures. Raman spectra changes observed during the lysis from "within" suggest alleged production of heat shock proteins are consistent with the occurring synthesis of phage-related proteins and are in good agreement with the calculated potential contribution of the above proteins to the Raman spectra. It was also established and validated that the contribution of cellular DNA to the Raman spectra of bacterial cells is negligible compared to RNA. The results open new venues for LTRS research and strongly suggest that LTRS has a great potential especially in investigation of real-time processes.

HPA axis exhaustion and regulatory T cell accumulation in patients with a functional somatic syndrome: recent view on the problem of Gulf War veterans.

The authors proceeded from the assumption that physical and mental symptoms of functional somatic syndromes (including those observed in Gulf War veterans) are based on both underactivity of hypothalamic-pituitary-adrenal (HPA) axis and excessive accumulation of regulatory T cells (Tregs). Permanent psychogenic stress coupled with high antigen loading leads to gradual depletion of HPA axis, which is manifested by the reduction of stress-induced cortisol response. Under stress hormone deficiency, Tregs begin to play a principal role in anti-inflammatory mechanisms and each new pro-inflammatory stimulus increases their number. Superfluous accumulation of active Tregs results in malfunction of Th1 cells in the brain that leads to the appearance of neurodegeneration foci, which seems to be an anatomic substance for various cognitive and psychological symptoms. New approaches to the treatment of such conditions are also discussed.

Alkylating drugs applied in non-cytotoxic doses as a novel compounds targeting inflammatory signal pathway.

Alkylating drugs (ADs) belonging to the nitrogen mustard family are commonly used as cytostatic and immunosuppressive agents. Our previous in vitro studies demonstrated that in the case of gradual dose decrease, the number of targets for alkylation in the cell is also reduced and the drug switches from brutal cytostatic to cell growth modifier. At doses of 0.3 microg/ml and lower, the effects of ADs are no longer associated with DNA damage or stress/MAPK pathways activation. Instead, the disruption of signal transduction by the IL-2beta and/or TNFalpha cell surface receptors is observed. As a result, ADs in the doses 100-fold lower than cytostatic ones are capable to modify lymphocyte activity including the activity of regulatory T cells. We hypothesized that ADs may have a beneficial effect in the treatment of inflammatory diseases. Indeed, the application of non-cytotoxic doses of an AD melphalan reduces the severity of murine experimental colitis. Daily administration of melphalan (25 microg/kg body weight) markedly reduced the severity of DSS-colitis as determined by clinical and histological criteria. Moreover, the beneficial effect of melphalan was also shown in asthmatic patients. In 60% of these patients histological and ultrastructural signs of bronchial epithelium regeneration were also revealed. Thus, ADs at non-cytotoxic concentrations exert beneficial effect both in acute and chronic inflammatory diseases. Such anti-inflammatory activity is thought to be due to blocking of signal transduction through various cell surface receptor including IL-2R and TNFR. Consequently different steps of inflammatory cascade turn out to be inhibited.

An extended -10 promoter alone directs transcription of the DpnII operon of Streptococcus pneumoniae.

The genetic cassette encoding the DpnII restriction-modification system of Streptococcus pneumoniae gave transcription products of approximately 2.7 and 1.8 kilobases. The larger, mRNA1, covered both of the methylase genes, dpnM and dpnA, and the endonuclease gene dpnB; the smaller, mRNA2, covered only the dpnA and dpnB genes. Transcription of mRNA1 was shown to begin at the translation start site for dpnM, thereby producing an mRNA without any apparent ribosome-binding site for translation of the DpnM methylase. The promoter for mRNA1 was shown by base substitution and deletion analysis to consist of an extended -10 site, TaTGgTATAAT, with no required -35 site. A possible promoter further upstream with close matches to a -35 site and a nonextended -10 site was not used. A survey of 36 proven and putative promoters used by S. pneumoniae revealed that 61% of them contained the full -10 extension, although, other than the dpnM promoter, they matched at a -35 site, as well. It appears that, unlike those found in Escherichia coli, S. pneumoniae promoters frequently require an extended -10 site, and such a site can function naturally without a -35 site.

Possible regulation of DNA methyltransferase expression by RNA processing in Streptococcus pneumoniae.

Atypical ribosome-binding sites lacking Shine-Dalgarno sequences appear to be used for translation of the DpnM and DpnA DNA methyltransferases of the DpnII restriction system. Preliminary results indicate that the 5'-endpoints of DpnII system mRNAs result from degradation of the original transcript. These tentative findings serve as the basis for a possible regulatory model that would accommodate the DpnII cassette either as a single copy in the chromosome or on a multicopy plasmid.

Nucleic acid transfer through cell membranes: towards the underlying mechanisms.

Various cases of DNA (RNA) transfer through membranes of living cells are reviewed. They are classified into two major categories: those which occur in Nature (natural transfer) and those imposed by various physical and chemical treatments of cells (induced transfer). Among the examples of natural transfer surveyed are the transfer during bacterial conjugation, genetic transformation, viral infection of bacteria, and nuclear membrane trafficking. Consideration of the induced transfer is focused on the two methods most widely used at present to introduce foreign genetic information into pro- and eukaryotic cells: Ca2+ (and some other divalent cations)-induced and calcium phosphate-induced transfer, and transfer during electroporation of cells. Emphasis is made on the underlying mechanisms of transfer, or rather on what is currently known about them. Energetic aspects of transfer are also discussed and different tentative models of transfer are presented.

Escherichia coli membranes during electrotransformation: an electron microscopy study.

Structural changes undergone by Escherichia coli cell envelope membranes under the conditions of electrically induced gene (DNA) transfer (exponential pulse of about 13 kV/cm, tau = 5 ms) were studied by freeze-fracture electron microscopy. Special device similar to that of Stenger and Hui [1986) J. Membr. Biol. 93, 43-53), that allowed cryofixation of samples almost simultaneously with application of electric pulse, was employed to examine the cells within a short time (less than or equal to 1 s) after the pulse. Extensive blebbing of cells was observed immediately after the pulse. At later times (30-40 s after the pulse) blebbing was not detected, instead infrequent cellular membrane fusion and formation of large membrane 'opening' or pores were observed. An attempt to relate the observed membrane changes with cellular viability and permeability to exogenous DNA failed. Challenge of cells with a plasmid DNA 10 s after the pulse application resulted in a dramatic loss (at least four orders of magnitude) of the number of transformants compared to cells pulsed in the presence of DNA. On the other hand the results on additional pulsing of cell prior to the main electrotransformation procedure suggested that the life-time of membrane defects is at least no less than 2 min. Possible ways to reconcile the results are suggested.

Plasmid transformation of Bacillus cereus on cellophane membranes.

A simple approach to test the ability of bacteria to undergo natural genetic transformation is suggested. The basic feature of the approach is the cultivation of bacterial cells in the presence of exogenous (plasmid) DNA on cellophane membranes placed successively on nutrient and selective agar. Using this approach the ability of Bacillus cereus for "natural" genetic transformation was detected. Transformation frequencies varied from 10(-8) to 10(-6).

Lipid polymorphism of model and cellular membranes as revealed by electron microscopy.

This review surveys the current state of knowledge relating to lipid polymorphism within both model lipid membrane and cellular membrane systems. Emphasis is placed upon the contribution of data obtained by transmission electron microscopy of freeze-fractured specimens. Some consideration is also given to the other important methods for the study of lipid polymorphism, namely X-ray diffraction and NMR spectroscopy. A detailed account of the different phases found in lipid mixtures within model membranes (bilayer, cubic or tetragonal, tubular or hexagonal) provides the background to the understanding of the factors involved in polymorphic phase transitions. The sequential steps involved in lipid polymorphism are defined from electron microscopical data and are related to the structural changes that can be detected within biological membranes. It is proposed that the fine structural changes detected at the initial stages of polymorphic transition in vivo may be highly relevant in relation to membrane fusion events, to the formation of tight junctions, and even to physiological transport processes. Since the later stages of polymorphic transition generally destroy the permeability barrier of model and cellular membranes, extensive rather than localized phase transition of the lipid bilayer is not at the moment considered to be compatible with cellular viability.

The in vivo formation of nonbilayer lipid phase in E. coli membranes during the development of Ca2-dependent competence.

31P-NMR studies on E. coli cells reveal the in vivo formation of nonbilayer lipid structures, presumably of H11 phase, during Ca2-dependent competence induction. The data suggest the involvement of these structures in the exogenous DNA transfer into the cells during genetic transformation and transfection. The suggestion is supported by in vitro experiments in which the liposomes composed of different phospholipid species bind 14C-DNA in DNase and wash resistant form in conditions promoting the hexagonal phase formation.

Can Ca2+-dependent competence be repeatedly induced in the same Escherichia coli cells?

With the help of devised multicycle consecutive transformation (MCT) it is shown that Ca2+-dependent competence can be repeatedly induced in the same population of Escherichia coli cells. The same fraction of cells is induced to competence and transformed during MCT. In contrast to the results on classical transformation with mixed DNA preparations, no double transformants are observed in MCT. The competent cells and transformants are found to be more fragile than nontransformed cells. The latter are represented presumably by the cells that have not absorbed exogenous plasmid DNA. The results suggest that there is strong interference between plasmid DNAs during MCT, and that the presence of exogenous DNA makes the cells more sensitive to the apparently harmful procedure of repeated competence induction.

Effect of metabolic inhibitors on entry of exogenous deoxyribonucleic acid into Ca2+-treated Escherichia coli cells.

The effect of various metabolic inhibitors (carbonylcyanid-m-chlorophenylhydrazone, nigericin, valinomycin, dicyclocarbodiimide, arsenate, NaF, etc.) and lipid-soluble synthetic ions (tetraphenylphosphonium bromide and tetraphenylboron sodium) on deoxyribonucleic acid (DNA) entry during transformation of Ca2+-treated Escherichia coli cells with plasmid DNA and on cell viability was investigated. In contrast to intact cells, Ca2+-treated E. coli cells were permeable to nigericin, valinomycin, and the other drugs tested. The inhibitors differentially affected [14C]proline active transport, and whereas some drugs inhibited transformation, the effects did not correlate with the effects on transport. The most potent inhibitors of transformation were nigericin, dicyclocarbodiimide, and tetraphenylboron sodium. Carbonylcyanid-m-chlorophenylhydrazone, tetraphenylphosphonium bromide, and valinomycin were relatively inactive. Tetraphenylboron sodium- and nigericin-treated cells bound were plasmid [14C]DNA in the deoxyribonuclease-resistant form than the control and other sample cells. Nevertheless, te penetration of exogenous plasmid DNA into the cell was greatly reduced, at least in case of nigericin. Unlike the other drugs, nigericin and dicyclocarbodiimide drastically affected the cell viability, the former within very short times of interaction. It is concluded that proton motive force does not play any significant role in DNA entry into Ca2+-treated E. coli cells. The results also suggest that adenosine 5'-triphosphate is not required for DNA entry either. The inhibitory effect of certain drugs is discussed in terms of structural perturbations induced by the drugs in cell envelope membranes.

Proton conductor vs. cold in induction of Ca(2+)-dependent competence in Escherichia coli.

In order to elucidate the molecular mechanisms of Ca(2+)-dependent competence in gram-negative bacteria an attempt was made to induce the competence at room temperature in presence of a proton conductor, carbonylcyanide-m-chlorophenylhydrazone (CCCP). Escherichia coli K12 cells treated with Ca2+ at 25 degrees or 37 degrees C in presence of CCCP became permeable for transforming plasmid and transfecting DNAs and DNA-binding antibiotic actinomycin C (AmC) and rubomycin (Rm) at room temperature. The efficiencies of transformation and transfection, however, were by 1-3 orders of magnitude lower compared to cells, treated with Ca2+ at 0 degree C, though both recipients did not differ significantly in their susceptibility to AmC and Rm. Possible mechanisms of Ca2+ action in both recipient systems are discussed in terms of molecular interactions.

Enhanced uptake of donor DNA by Ca2+ treated Escherichia coli cells.

The first steps in E. coli transformation have been studied. It was found that the cells attain the ability of enhanced uptake of donor exogenous DNA upon Ca2+ treatment. At saturating DNA concentrations the cells were capable to take up irreversibly about 6-10(8) dalton of donor DNA per cell (if to assume that all the cells are competent). About 75% of this DNA was found to be attached to the cytoplasmic membrane. Employing Poisson approximation for distribution of the donor-marker DNA molecules on recipient cells it was found that the efficiency of a marker recombination and expression is about 2-10(-5). The first steps in E. coli transformation have been studied. It was found that the cells attain the ability of enhanced uptake of donor 0exogenous