Ultrasound Mediated Delivery of Compounds into Petunia Protoplasts and Cells

The effects of ultrasound on uptake of foreign substance by Petunia protoplasts were studied using calceln (3, 6-dihydroxy-2,4- bis-[N, N-di (carboxymethyl)—aminoethyl fluoran] and DNA. Neither the protoplasts nor cells took up the calcein, but treatment with ultrasound facilitated the uptake. Pronounced stimulation of uptake was observed with protoplasts but only moderate stimulation was observed with the cells. The ultrasound uptake was dependent on sound frequency, temperature and duration of treatment. Uptake increased with treatment time but prolong treatment caused the rupture of protoplasts. Ultrasound also facilitated uptake of DNA by cells of Petunia. In this study the plasmid pBI 121.2 carrying a reporter, β-glucuronidase (GUS)’gene was used. Transient expression of this gene was an indicator of DNA uptake. Survived protoplasts and cells subjected to ultrasound treatment divided and formed microcalli in agarose medium.     

Mechanism of partial adaptation in airway smooth muscle after a step change in length.

The phenomenon of length adaptation in airway smooth muscle (ASM) is well documented; however, the underlying mechanism is less clear. Evidence to date suggests that the adaptation involves reassembly of contractile filaments, leading to reconfiguration of the actin filament lattice and polymerization or depolymerization of the myosin filaments within the lattice. The time courses for these events are unknown. To gain insights into the adaptation process, we examined ASM mechanical properties and ultrastructural changes during adaptation. Step changes in length were applied to isolated bundles of ASM cells; changes in force, shortening velocity, and myosin filament mass were then quantified. A greater decrease in force was found following an acute decrease in length, compared with that of an acute increase in length. A decrease in myosin filament mass was also found with an acute decrease in length. The shortening velocity measured immediately after the length change was the same as that measured after the muscle had fully adapted to the new length. These observations can be explained by a model in which partial adaptation of the muscle leads to an intermediate state in which reconfiguration of the myofilament lattice occurred rapidly, followed by a relatively slow process of polymerization of myosin filaments within the lattice. The partially adapted intermediate state is perhaps more physiologically relevant than the fully adapted state seen under static conditions, and it simulates a more realistic behavior for ASM in vivo.     

The potential application of chlorin e6-polyvinylpyrrolidone formulation in photodynamic therapy.

Much research has been focused on developing effective drug delivery systems for the preparation of chlorins as potential photosensitizers for PDT. This report describes the evaluation of a new water-soluble formulation of chlorin e6 consisting of a complex of trisodium salt chlorin e6 and polyvinylpyrrolidone (Ce6-PVP) for application in photodynamic therapy (PDT) with 2 specific aims: (i) to investigate its fluorescence kinetics in skin, normal and tumor tissue after intravenous administration, and (ii) to investigate its PDT efficacy. Our results demonstrate that this new formulation possesses photosensitizing properties with rapid accumulation in tumor tissue observed within 1 h after intravenous administration. Although high selectivity in tumor tissue was found between the period of 3 and 6 h, the efficacy of Ce6-PVP mediated PDT was best at 1 h drug-light interval. It is suggested that, the extent of tumor necrosis post PDT is dependent on the plasma concentration of Ce6-PVP, implying a vascular mediated cell death mechanism. A faster clearance rate of Ce6-PVP from the skin of nude mice was observed compared to Ce6. The new formulation of Ce6-PVP seems to show promise as an effective therapeutic agent.     

Synthesis of teichoic acids. VII. Synthesis of teichoic acids during spore germination

The synthesis of teichoic acids has been examined during germination in Bacillus licheniformis ATCC 9945 and in B. subtilis W-23. Teichoic acids are absent from the spores of both organisms. B. licheniformis spores lack the enzymes responsible for teichoic acid synthesis. The appearance of these enzymes during germination is correlated with the appearance of teichoic acids in the cell. The appearance of teichoic acid-synthesizing enzymes and of teichoic acids in the cell are inhibited by the addition of chloramphenicol to the germination medium. In B. subtilis W-23 the situation is similar for the synthesis of polyribitolphosphate. The synthesis of glucosyl polyribitolphosphate is only partially inhibited by chloramphenicol, puromycin, and penicillin, and uridine diphosphate-d-glucose polyribitol-phosphate glucosyl transferase can be demonstrated in spores. The possible implications of some of these observations are discussed.     

Estimation of the number of viable particles of Histoplasma capsulatum in soil

Summary Serial dilutions of suspensions of soil samples positive forH. capsulatum were made and injected intravenously into mice. The dilution producing infection in 50 % of the mice injected (ID₅₀) was determined for each sample and provided a measure for quantitative comparisons. A known number of viable particles ofH. capsulatum was added to soil, and serial dilutions were made of the suspension and injected into mice to determine that dilution containing an ID₅₀. One ID₅₀ was calculated to contain 1.6 viable particles ofH. capsulatum per ml of inoculum. With the assumption that one ID₅₀ of unknown samples contained 1.6 viable particles per ml inoculum, the total number of viable particles per gram of soil in several sites was calculated. The total number of viable particles ofH. capsulatum per gram of soil in different sites ranged from 101 to 201,900, almost a two thousandfold difference. Now that the number of viable particles ofH. capsulatum in positive sites can be determined, it may be possible to determine the concentration of particles necessary to make sites significant sources of infection.From the Ecological Investigations Program, National Communicable Disease Center, Bureau of Disease Prevention and Environmental Control, Public Health Service, U.S. Department of Health, Education, and Welfare, Kansas City, Kansas.Presented in part at the annual meeting of the American Society for Microbiology, New York, N.Y., April 30-May 4, 1967.     

Somatic embryogenesis in cell suspension culture of carnation (Dianthus caryophyllus L.)

Somatic embryogenesis and plantlet formation were obtained from 60–75 day old cell cultures of carnation. Callus was generated on MS basal medium supplemented with 2,4-dichchlorophenoxy acetic acid (2,4-D). Removal of 2,4-D during subsequent subculturing of cell suspensions resulted in formation of embroids. These somatic embryos originated from single cells and their early development proceeded normally with clearly defined apical and root meristems. Some embryos developed into plants and were acclimatized to ex vitro conditions.Abbreviations BAP 6-benzylaminopurine - Kinetin 6-furfurylamino purine - 2,4-D 2,4-dichlorophenoxy acetic acid - MS Murashige and Skoog     

The 1952 Outbreak of Encephalitis in California (A Symposium): LABORATORY METHODS FOR ETIOLOGIC DIAGNOSIS

The general procedures used in the diagnosis of neurotropic viral diseases are outlined and are discussed with specific reference to western equine encephalitis.Cerebrospinal fluid is considered practically worthless as a starting material, in attempts to isolate the causal agent. The material of choice in attempting to recover the virus is central nervous system tissue, available only in instances of fatal infection. In the usual case, the diagnosis depends upon serologic or immunologic methods. These methods are aimed at detecting the presence of specific antibodies and of increases in the content of antibodies in the blood during the course of the illness.The in vitro complement fixation test is considered a better diagnostic tool than the in vivo neutralization test, since rises in titer are more readily detectable by the former technique than by the latter.     

Comparison of biophysical properties characterized for microtissues cultured using microencapsulation and liquid crystal based 3D cell culture techniques

Growing three dimensional (3D) cells is an emerging research in tissue engineering. Biophysical properties of the 3D cells regulate the cells growth, drug diffusion dynamics and gene expressions. Scaffold based or scaffoldless techniques for 3D cell cultures are rarely being compared in terms of the physical features of the microtissues produced. The biophysical properties of the microtissues cultured using scaffold based microencapsulation by flicking and scaffoldless liquid crystal (LC) based techniques were characterized. Flicking technique produced high yield and highly reproducible microtissues of keratinocyte cell lines in alginate microcapsules at approximately 350 ± 12 pieces per culture. However, microtissues grown on the LC substrates yielded at lower quantity of 58 ± 21 pieces per culture. The sizes of the microtissues produced using alginate microcapsules and LC substrates were 250 ± 25 μm and 141 ± 70 μm, respectively. In both techniques, cells remodeled into microtissues via different growth phases and showed good integrity of cells in field-emission scanning microscopy (FE-SEM). Microencapsulation packed the cells in alginate scaffolds of polysaccharides with limited spaces for motility. Whereas, LC substrates allowed the cells to migrate and self-stacking into multilayered structures as revealed by the nuclei stainings. The cells cultured using both techniques were found viable based on the live and dead cell stainings. Stained histological sections showed that both techniques produced cell models that closely replicate the intrinsic physiological conditions. Alginate microcapsulation and LC based techniques produced microtissues containing similar bio-macromolecules but they did not alter the main absorption bands of microtissues as revealed by the Fourier transform infrared spectroscopy. Cell growth, structural organization, morphology and surface structures for 3D microtissues cultured using both techniques appeared to be different and might be suitable for different applications.     

In vivo therapeutic applications of cell spheroids

Spheroids are increasingly being employed to answer a wide range of clinical and biomedical inquiries ranging from pharmacology to disease pathophysiology, with the ultimate goal of using spheroids for tissue engineering and regeneration. When compared to traditional two-dimensional cell culture, spheroids have the advantage of better replicating the 3D extracellular microenvironment and its associated growth factors and signaling cascades. As knowledge about the preparation and maintenance of spheroids has improved, there has been a plethora of translational experiments investigating in vivo implantation of spheroids into various animal models studying tissue regeneration.We review methods for spheroid delivery and how they have been utilized in tissue engineering experiments. We break down efforts in this field by organ systems, discussing applications of spheroids to various animal models of disease processes and their potential clinical implications. These breakthroughs have been made possible by advancements in spheroid formation, in vivo delivery and assessment. There is unexplored potential and room for further research and development in spheroid-based tissue engineering approaches. Regenerative medicine and other clinical applications ensure this exciting area of research remains relevant for patient care.