Mitogen response and cell cycle kinetics of Swiss 3T3 cells in defined medium: differences from human fibroblasts and effects of cell density

The mitogen requirement and proliferative response of Swiss 3T3 cells in serum-free, chemically defined culture medium were compared with those of early-passage human diploid fibroblasts. The effects of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), insulin, transferrin, and dexamethasone on cell-cycle parameters were measured using 5'-bromo-deoxyuridine-Hoechst flow cytometry. Swiss 3T3 cells differ from human fibroblasts in several ways: (1) Swiss 3T3 cells showed a much higher dependence on PDGF than human fibroblasts; the growth of the latter, but not of the former, could be stimulated by the combination of EGF, insulin, and dexamethasone to the full extent of that when PDGF was present; (2) in the absence of PDGF, insulin was an absolute requirement for Swiss 3T3 cells to initiate DNA synthesis, while a substantial proportion of human fibroblasts could enter DNA synthesis without exogenous insulin or IGF-I; and (3) in the absence of PDGF, increasing insulin concentration increased the cycling fraction of Swiss 3T3 cells without an appreciable effect on the rate of cell exit from G0/G1, while under similar culture conditions, insulin showed its major effect on regulation of the G1 exit rate of human fibroblasts, without much effect on the cycling fraction. In addition, the proliferative response of high-density versus low-density, arrested Swiss 3T3 cells showed that the interaction of mitogens varied with cell density. At high cell density, the PDGF requirement was consistent with the "competence/progression" cell-cycle model. This growth response was not seen, however, when cells were plated at low density.     

Scanning electron microscopy ofMycoplasma pneumoniae on the membrane of individual ciliated tracheal cells

Summary Cell monolayer cultures were prepared from hamster tracheal explants by a collagenase exposure and subsequent incubation in Waymouth’s MAB 87/3 medium. The epithelial outgrowth occurred on glass cover slips. Cilia on the monolayers continued to beat normally after the “parent” explant was removed. Monolayer cultures infected withMycoplasma pneumoniae had significant amounts of attachment. A morphological analysis of the attachment was conducted with scanning electron microscopy. Clusters, cocci, and filaments ofM. pneumoniae all attached to the epithelial cells, but the filaments were especially common. Mycoplasmas were seen in association with both ciliated and nonciliated cell membranes. On ciliated cells, mycoplasmas were on the ciliary strands and on the cell membrane. When located immediately adjacent to or in between cilia, mycoplasmas were oriented vertically with the constricted attachment tip oriented down toward the host cell membrane. When located more than a micron away from the ciliary fibers, mycoplasmas lay horizontally along the epithelial cell membrane. The photographic data suggest that clusters or “sperules” of mycoplasmas may liberate individual mycoplasmas that attach to the cell membrane. It appears that the receptor sites forM. pneumoniae are rather uniformly distributed along the ciliated cell membrane, and are not restricted to the interciliary areas. Electron microscopy was done with the cooperation of Dr. R. Macleod and the staff of the Center for Electron Microscopy at the University of Illinois. Critical editorial review was provided by C. Dayton. This investigation was supported in part by grants to M. G. G. from the National Institute of Allergy and Infectious Diseases (AI 12559) and the National Heart, Lung, and Blood Institute (HL 23806), Bethesda, Maryland.     

Random recirculation of small T lymphocytes from thoracic duct lymph in the mouse

The migration of ⁵¹Cr-labeled nylon-wool separated mouse thoracic duct T cells has been followed in order to determine whether there is a circulation of small (nondividing) T cells through the small intestine. Approximately 6% of the injected dose of T-TDL localized in the small intestine (minus Peyer's patches). Experiments revealed that this gut-localizing cell population consisted almost entirely, if not exclusively, of lymphoblasts present in mouse T-TDL. When lymphoblasts and small lymphocytes from mouse T-TDL were separated by velocity sedimentation, and the migration of separated fractions was studied, we found large cells (66% blasts) migrated well to the gut but poorly to the lymph nodes, whereas small cells (2% blasts) showed minimal migration to the gut but localized randomly in lymph nodes and spleen. The in vivo distribution of small cells from T-TDL was similar to that of T-PLN. Furthermore, the recirculatory patterns of both ⁵¹Cr-labeled T-TDL and T-PLN were found to be identical as accessed by their rate of recovery in the thoracic duct lymph of recipient mice. These results support the notion that the vast majority of T-TDL and T-PLN are part of a common pool of recirculating T cells which recirculate randomly through lymph nodes and spleen and not the small intestine.     

Cytochemistry and freeze-fracture of membranes isolated from the electrocyte of Electrophorus electricus (L.)

Summary Membranes were isolated from the main electric organ of Electrophorus electricus and studied by means of cytochemistry and freezefracture. The membrane fractions consisted of vesicles inside-in as determined by localization of anionic sites using colloidal iron and cationized ferritin particles. The anionic sites were not homogeneously distributed on the surface of the vesicle. Freeze-fracture showed the presence of intramembranous particles associated with either protoplasmic (P) or extracellular (E) faces of the membrane. Regions of the membrane without particles were observed. The results are discussed in relation to the existence of association between intramembranous particles and membrane receptors.For all correspondence     

Gain and loss of fluid in metamorphosing larvae of Manduca sexta

Larvae of Manduca sexta, (Sphingidae) increase their weight approx. 50% just before pupation and then secrete this fluid during the formation of their burrows. Time-lapse cinematography indicates that the fluid is ejected into the walls of the final burrow. It may offer some mechanical support; it is not particularly repellent to ants or mice, and it contains only small amounts of the alkaloids ingested from its preferred food plants. Comparison to other species indicates that the gain and loss of water is associated with burrowing behaviour; the fluid appears to be used in providing hydraulic pressure for burrowing, in forming and cementing the pupal chamber, and in acting as a CO₂ trap underground. The secretion is a hypertonic, highly alkaline solution containing KHCO₃ and small amounts of Na⁺, Ca²⁺, Mg²⁺, PO₄^?3 and some proteins. Haemolymph levels of K⁺ decrease, and those of Ca²⁺ increase, during the secretory phase. When radioactive calcium is injected into mature larvae, it appears promptly in the secretion. If however, the injection is given more than 24 hr before the animal begins secreting, then the calcium is bound to haemolymph protein and does not appear in the secretion.     

A redox trap to augment the intein toolbox

The unregulated activity of inteins during expression and consequent side reactions during work‐up limits their widespread use in biotechnology and chemical biology. Therefore, we exploited a mechanism‐based approach to regulate intein autocatalysis for biotechnological application. The system, inspired by our previous structural studies, is based on reversible trapping of the intein's catalytic cysteine residue through a disulfide bond. Using standard mutagenesis, the disulfide trap can be implemented to impart redox control over different inteins and for a variety of applications both in vitro and in Escherichia coli. Thereby, we first enhanced the output for bioconjugation in intein‐mediated protein ligation, also referred to as expressed protein ligation, where precursor recovery and product yield were augmented fourfold to sixfold. Second, in bioseparation experiments, the redox trap boosted precursor recovery and product yield twofold. Finally, the disulfide‐trap intein technology stimulated development of a novel bacterial redox sensor. This sensor reliably identified hyperoxic E. coli harboring mutations that disrupt the reductive pathways for thioredoxin and glutathione, against a background of wild‐type cells. Biotechnol. Bioeng. 2013; 110: 1565–1573. © 2012 Wiley Periodicals, Inc.     

Successful use of a novel lux® i-Amylose-1 chiral column for enantioseparation of “legal highs” by HPLC

Bath salts, fumigations, cleaners and air fresheners, behind these terms substances are hidden, which count as “Legal Highs”. These fancy names are used to pretend Legal Highs as harmless compounds, to circumvent legal regulations for marketing as well as to increase the sales. Besides classic illicit drugs of synthetic origin such as amphetamines, cocaine and MDMA, the trade of these compounds, also known as new psychoactive substances (NPS), is not uncommon today. In many countries, NPS are still not subject to drug control. Among them, there are stimulants such as new amphetamine derivatives or cathinones, which possess a chiral centre. Little is known about the fact that the two possible enantiomers may differ in their pharmacological effect. The aim of this study was to test a novel HPLC column for the enantioseparation of a set of 112 NPS coming from different chemical groups and collected by internet purchases during the years 2010–2018. The CSP, namely Lux® 5 μm i-Amylose-1, LC Column 250 x 4.6 mm, was run in normal phase mode under isocratic conditions, UV detection was performed at 245 nm and 230 nm, injection volume was 10 μl and flow rate was 1 ml/min. With a mobile phase consisting of n-hexane/isopropanol/diethylamine (90:10:0.1), herein, 79 NPS were resolved into their enantiomers successfully, for 37 of them baseline resolution was achieved. After increase of lipophily of the mobile phase to 99:1:0.1, another 27 compounds were baseline separated. It was found that all separated NPS are traded as racemic compounds.