Cytoplasmic molecular delivery with shock waves: importance of impulse

Cell permeabilization using shock waves may be a way of introducing macromolecules and small polar molecules into the cytoplasm, and may have applications in gene therapy and anticancer drug delivery. The pressure profile of a shock wave indicates its energy content, and shock-wave propagation in tissue is associated with cellular displacement, leading to the development of cell deformation. In the present study, three different shock-wave sources were investigated; argon fluoride excimer laser, ruby laser, and shock tube. The duration of the pressure pulse of the shock tube was 100 times longer than the lasers. The uptake of two fluorophores, calcein (molecular weight: 622) and fluorescein isothiocyanate-dextran (molecular weight: 71,600), into HL-60 human promyelocytic leukemia cells was investigated. The intracellular fluorescence was measured by a spectrofluorometer, and the cells were examined by confocal fluorescence microscopy. A single shock wave generated by the shock tube delivered both fluorophores into approximately 50% of the cells (p < 0.01), whereas shock waves from the lasers did not. The cell survival fraction was >0.95. Confocal microscopy showed that, in the case of calcein, there was a uniform fluorescence throughout the cell, whereas, in the case of FITC-dextran, the fluorescence was sometimes in the nucleus and at other times not. We conclude that the impulse of the shock wave (i.e., the pressure integrated over time), rather than the peak pressure, was a dominant factor for causing fluorophore uptake into living cells, and that shock waves might have changed the permeability of the nuclear membrane and transferred molecules directly into the nucleus.     

A case of spontaneous coronary artery disease regression

Coronary angiographic trials have demonstrated that lowering cholesterol can slow the progression of atherosclerosis, limit the formation of new lesions and enhance atherosclerotic regression together with reducing the incidence of clinical events (Waters D, 1996). Spontaneous regression of coronary atherosclerotic lesions is rare. We report the case of a patient with a severe within-stent restenotic lesion whose coronary disease spontaneously regressed 12 months after initial diagnosis, allowing for medical treatment of symptoms rather than repeated intervention. (Int J Cardiovasc Interventions 1999; 2: 121-123)     

Lithium stimulation of diffusion chamber colony growth is mediated by factors other than colony-stimulating factor

Lithium is a recognized, potent stimulator of granulopoiesis. The present study used the model of clonal growth of granulopoietic precursors in diffusion chambers to investigate the relevance of certain colony-stimulating factors to lithium stimulation in vivo. In this system, lithium stimulation of granulopoiesis could not be attributed to changes in serum or chamber fluid colony-stimulating factor levels. Antibody to colony-stimulating factor-1 administered during culture markedly reduced morphologic expression of colonies in control and lithium-pretreated host mice, yet subculture of chamber contents revealed that lithium stimulation of a granulopoietic progenitor, perhaps of primitive potentiality, had nevertheless occurred. Therefore, we hypothesize that lithium acts in an indirect, hormonal fashion and that these colony-stimulating factors, while necessary for morphologic expression, play no role in the stimulatory effect. This hypothesis raises the possibility that lithium in combination with recombinant colony-stimulating factors may result in clinically effective synergistic stimulation of granulopoiesis.     

Factors influencing the release of proteins by cultured schwann cells

Cultured rat schwann cells grown in association with sensory neurons when labeled with [(3)H]leucinem, [(3)H]glucosamine, or [(35)S]methionine release labeled polypeptides into the culture medium. Analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of the culture medium reveals a reproducible pattern of more than 20 polypeptides with molecular weights ranging from 15,000 to more than 250,000. Five major polypeptides (apparent molecular weights 225,000, 210,000, 90,000, 66,000, 50,000, and 40,000) account for approximately 40 percent of the leucine or methionine radioactivity in medium polypeptide. Schwann cells grown in a serum-free defined medium, in which schwann cells do not relate normally to axons, release approximately four times less labeled medium polypeptides tha cultures grown in medium supplemented with serum and chick embryo extract. In addition, there is a qualitative difference in the pattern of medium polypeptides resolved by SDS-PAGE, so that a single polypeptide (mol wt 40,000) accounts for nearly all of the label in medium polypeptides. Switching of cultures grown in defined medium to supplemented medium for 2 d results in a fourfold increase in the amount of labeled polypeptides appearing in the culture medium, and a return to the normal pattern of medium polypeptides appearing in the culture medium, and a return to the normal pattern of medium polypeptides as resolved by SDS-PAGE. This change in the pattern of polypeptides release by schwann cells is accompanied by changes in the association between schwann cells and axons. An early step in the establishment of normal axon-schwann cell relations appears to be an inward migration of schwann cells into axonal bundles and spreading of schwann cells along neurites. These changes are evident within 48 h after medium shift. Our results thus suggest that the release of proteins by schwann cells may be important for the development of normal axonal ensheathment.     

Resonance Raman studies of the primary photochemical event in visual pigments.

Resonance Raman multicomponent spectra of bovine rhodopsin, isorhodopsin, and bathorhodopsin have been obtained at low temperature. Application of the double beam "pump-probe" technique allows us to extract a complete bathorhodopsin spectrum from the mixture in both protonated and deuterated media. Our results show that the Schiff base of bathorhodopsin is fully protonated and that the extent of protonation is unaffected by its photochemical formation from either rhodopsin or isorhodopsin. The Raman spectrum of bathorhodopsin is significantly different than that of either parent pigment, thus supporting the notion that a geometric change in the chromophore is an important component of the primary photochemical event in vision. A normal mode analysis is carried out with particular attention devoted to the factors that determine the frequency of the C=N stretching vibration. We find that the increased frequency of this mode in protonated relative to unprotonated Schiff bases is due to coupling between C=N stretching and C=N-H bending motions, and the shift observed upon deuteration of the Schiff base can also be understood in these terms. Various models for the primary event are discussed in light of our experimental and theoretical results.     

The relationship between codon boundaries and multiple reading-frame preferences: coding organization of bacterial insertion sequences

Theoretical considerations have shown that the five possible overlapping reading-frame configurations differ significantly in their coding flexibility and thus in their information content (Siegel and Fitch 1980; Smith and Waterman 1980). Contrary to expectation, the overlapping frame configuration allowing the greatest coding flexibility is rarely seen, whereas one of the most constraining is common. We point out here that this overlapping reading-frame paradox and an observed but unexplained preference in coding regions for a pyrimidine-purine at codon boundaries (Shepherd 1981; Jones and Kafatos 1982; Smith et al. 1983) are intimately linked. The codon boundary preference, which may be related to translation efficiency or accuracy, places constraints on the evolution of overlapping coding regions. These considerations may help identify actual coding regions in DNA sequences. We have analyzed five sequenced (enteric) bacterial insertion sequences for codon boundary incidences and reading-frame configurations and find that they are consistent with these proposed constraints.      

Studies on the actin-binding protein HS1 in platelets

Background  

The platelet cytoskeleton mediates the dramatic change in platelet morphology that takes place upon activation and stabilizes thrombus formation. The Arp2/3 complex plays a vital role in these processes, providing the protrusive force for lamellipodia formation. The Arp2/3 complex is highly regulated by a number of actin-binding proteins including the haematopoietic-specific protein HS1 and its homologue cortactin. The present study investigates the role of HS1 in platelets using HS1^-/- mice.     

Retargeted delivery of adenoviral vectors through fibroblast growth factor receptors involves unique cellular pathways.

A major goal of gene therapy is to improve target specificity by delivering vectors through alternative cellular receptors. We previously reported that adenoviral vector delivery through basic fibroblast growth factor (FGF2) receptors enhances both cellular transduction and in vivo efficacy. We now present studies addressing the cellular pathways and mechanisms underlying these events. Cellular receptors for adenoviruses are not required for transduction by FGF2-retargeted vectors. Moreover, alpha(V) integrins can antagonize FGF2 retargeting, in contrast to their obligatory role in non-retargeted vector delivery. By contrast, high-affinity FGF receptors, which are overexpressed on potential tumor targets, are required for FGF2-retargeted transduction. Low-affinity heparan sulfate proteoglycan interactions, however, are not a prerequisite, in marked contrast to their obligatory role in FGF2 mitogenic signaling. By comparing receptor expression and ligand binding with transgene expression, we also demonstrate that FGF2 retargeting enhances transduction by mechanisms other than increasing the number of targeted cells. Rather, the use of alternative targeting ligands supports the conclusion that specific receptor interactions and intracellular events serve to enhance transgene expression. Together, these studies highlight the unique delivery and transduction pathways used by FGF2-retargeted adenoviruses, and help define the basis for their enhanced in vivo efficacy.     

<i>Trichoderma</i> spp. fostering growth on <i>Capsicum chinense</i> Jacq. seedlings and antagonistic against <i>Meloidogyne incognita</i>

Fourteen native strains of Trichoderma spp. from wildand agricultural pathosystems in the state of Yucatan, Mexico, with growth-promoting ability of Capsicum chinense Jacq. seedlings were evaluated and antagonistic effect of their filtrate against second-stage juveniles (J2) of Meloidogyne incognita. The strains Th05-02 and Th27-08 showed the best significant effects on plant hight variable increments 55.57 and 47.62%, theTh07-04 with 29.48% more root length, theTh02-01 and Th07-04 isolates increased from 48.71 to 84.61% in volume radical and 53.40% of total dry biomass. Statistical analysis (p≤0.001) of Th43 and Th43-13-14 filtrates caused 100% mortality at 24 and 48h. In the test of reversibility to 24 h after replacing the filtrates Th43-13, Th43-14, TH09-06 and TH20-07 by sterile distilled water, the J2 did not recover their viability, so they were considered as the best potential strains of Trichoderma spp. with antagonistic capacity in J2 of M.incognita.     

Picosecond kinetic absorption and fluorescence studies of bovine rhodopsin with a fixed 11-ene

A synthetic retinal having a fixed 11-cis geometry has been used to prepare a nonbleachable analogue of bovine rhodopsin. Marked differences in the picosecond absorption and fluorescence behavior of this analogue at room temperature, compared with that of natural rhodopsin, were observed. This not only indicates that the 11-cis to trans isomerization of the retinal moiety is the crucial primary event in the photolysis of rhodopsin, but also it establishes that this isomerization must occur on the picosecond time scale or faster.