A method for functional evaluation of caspase activation pathways in intact lymphoid cells using electroporation-mediated protein delivery and flow cytometric analysis.

The purpose of the study was to develop a rapid technique for determining the functional status of caspase activation pathways in intact lymphocytes. Proteins known to activate caspase-family cell death proteases (cytochrome c; granzyme-B; caspase-8) were introduced into human leukemia and lymphoma cell lines, as well as freshly isolated lymphocytes and leukemia cells, by electroporation. Fluorochrome-labeled proteins with a wide range of molecular weights (from 15 to 150 kDa) were used to evaluate electroporation efficiency by flow cytometry and to compare the efficiency of protein delivery using various electroporation conditions. Caspase activity was monitored using a cleavable, cell-permeable fluorogenic substrate. Conditions were identified for efficient delivery of proteins of +150 kDa into lymphoid cells. Caspase activation induced by various proteins was compared in normal and leukemic lymphocytic cells, revealing impaired caspase activation pathways in some malignant cells. We conclude that electroporation of apoptotic proteins into intact lymphoid cells can be used to contrast the status of various caspase activation pathways, thereby providing insights into the pathological defects in apoptosis regulation that exist in individual patient specimens.

Whole-column imaging capillary electrophoresis of proteins with a short capillary.

Whole-column imaging capillary electrophoresis with a short capillary is discussed. A short capillary (3-6 cm) coated with either fluorocarbon or polyacrylamide was used as a separation capillary. The whole capillary was illuminated with 280 nm light, and the transmitted light was monitored by a linear charge-coupled device (CCD). For the short capillary, hydrodynamic flow caused by a subtle height difference between the anodic and cathodic reservoirs affected the sample migration in the capillary greatly. Several sample injection methods, including use of a cross connection, sealing of the capillary ends with a gel, and use of a gel-filled capillary, have been discussed. The experimental results showed that the peak height decreased and peak width increased with the electromigration distance. Therefore, higher sensitivity was obtained in a short capillary rather than a long capillary. The whole-column imaging CE with the short capillary has been applied for the study of conjugation reactions of protein cytochrome c with sodium dodecyl sulfate (SDS) and the dye Congo Red. The method has also been used for in situ monitoring of the electrophoretic protein desorption process. Our technique is a unique tool for the study of protein binding reactions and the interaction between analyte and inner wall of the capillary.

Target-derived cardiotrophin-1 and insulin-like growth factor-I promote neurite growth and survival of developing oculomotor neurons.

Several trophic factors support the survival of developing motoneurons, but it is not known whether these factors act in a retrograde fashion from the motoneuron target muscle or are derived from other sources. Cardiotrophin-1 (CT-1) and the insulin-like growth factors (IGFs) are candidate target-derived motoneuron survival factors as both are expressed in muscle during naturally occurring motoneuron death and, applied systemically, support the survival of developing motoneurons. By using the embryonic chick oculomotor system, we show that CT-1 and IGF-I promote neurite outgrowth from E13-derived oculomotor explants and are retrogradely transported from muscle to nerve cell body in vivo, and injection of CT-1 or IGF-I into eye muscles increases motoneuron survival by 20 and 30%, respectively, as evidenced by calibrated stereological counting techniques. Pharmacological depletion of endogenous target-derived IGF-I in vivo reduces oculomotor neuron survival by up to 30% in a dose-dependent manner. These results significantly extend previous studies using systemic administration of trophic factors and are the first to demonstrate a target-derived retrograde mechanism of developing motoneuron survival factors.

Mitochondrial swelling and cytochrome c release: sensitivity to cyclosporin A and calcium.

The opening of mitochondrial membrane permeability transition (MPT) pores, which results in a cyclosporin A (CsA)-sensitive and Ca(2+)-dependent dissipation of the membrane potential (delta psi) and swelling (classical MPT), has been postulated to play an important role in the release of cytochrome c (Cyt.c) and also in apoptotic cell death. Recently, it has been reported that CsA-insensitive or Ca(2+)-independent MPT can be classified as non-classic MPT. Therefore, we studied the effects of apoptosis-inducing agents on mitochondrial functions with respect to their CsA-sensitivity and Ca(2+)-dependency. CsA-sensitive mitochondrial swelling, depolarization, and the release of Ca2+ and Cyt.c were induced by low concentrations of arachidonic acid, triiodothyronine (T3), or 6-hydroxdopamine but not by valinomycin and high concentrations of the fatty acid or T3. Fe2+/ADP and 2,2,-azobis-(2-amidinopropane) dihydrochloride (AAPH) induced swelling of mitochondria and the release of Ca2+ and Cyt.c were not coupled with depolarization or CsA-sensitivity while dibucaine-induced swelling occurred without depolarization, Cyt.c-release or by a CsA-sensitive mechanism. A protonophoric FCCP and SF-6847 induced depolarization and Ca(2+)-release occurred in a CsA-insensitive manner and failed to stimulate the release of Cyt.c. These results indicate that ambient conditions of mitochondria can greatly influence the state of membrane stability and that Cyt.c release may occur not only via a CsA-sensitive MPT but also by way of a CsA-insensitive membrane deterioration.

BDNF and NT3 extend the critical period for developmental climbing fibre plasticity.

The effect on neonatal brain plasticity of two neurotrophins, brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), was studied using the rat olivocerebellar projection as a model. Unilateral transection of climbing fibres (CFs) in the rat before postnatal day 7 induces reinnervation of the deafferented hemicerebellum, but this does not occur if the transection is performed after postnatal day 10. Eleven-day-old day rats underwent unilateral CF transection followed by neurotrophin injection into the denervated cerebellar cortex 24 h later. The exogenous neurotrophins induced CF reinnervation of the denervated hemicerebellum. However BDNF was more efficacious than NT-3. Thus two neurotrophins can extend the window of neonatal brain plasticity, therefore suggesting potential therapeutic use after brain trauma.

Endocytosis of ferritin and hemoglobin by the trabecular endocardium in swordtail, Xiphophorus helleri L. and platy, Xiphophorus maculatus L. (Poecilidae: Teleostei).

The cardiac atrium and ventricle of swordtail, Xiphophorus helleri L. and platy, Xiphophorus maculatus L., are spongious, consisting of muscle trabeculae covered by endocardial cells. The cardiac trabecular endocardium is able to take up and store large amounts of horse-spleen ferritin and bovine hemoglobin from the blood stream. No such uptake was registered in endocardial cells lining the cardiac valves, atrio-ventricular junction and ventriculo-bulbar junction. The trabecular endocardium in these species seems to be unable to accumulate latex beads or bovine myoglobin, cytochrome C and holotransferrin from the blood stream. It is proposed that the trabecular endocardium in these species is able to clear the blood stream of some types of waste macromolecules; i. e. this tissue may have a scavenger function. The present results indicate that the uptake of foreign ferritin in bony fish endocardium can be clearly demonstrated at the light microscopic level in deparaffined sections by means of acid ferrocyanide or Mallory solutions. A similar uptake of hemoglobin is demonstrated by means of Mallory stain.

Neonatally administered diethylstilbestrol retards the development of the blood-testis barrier in the rat.

Newborn rats were treated with 10 microg of diethylstilbestrol (DES) on alternate days from the 2nd to the 12th postnatal day, and the testes were sequentially examined up to 105 days of age by light, electron, and confocal laser microscopy. In control rats, spermatozoa and step 19 spermatids were observed in stage VIII seminiferous tubules at 56 days of age. Spermatogenic cells in DES-treated rats differentiated normally from birth until 21 days of age, after which differentiation continued only to the pachytene-spermatocyte stage. From this age onward, spermatogenic cells older than pachytene spermatocytes were not found until 56 days of age. After this point, the cells resumed differentiation and finally became spermatozoa by 91 days of age; that is, 35 days later than control rats. Electron and confocal laser microscopy showed that in the normal rat, the formation of the ectoplasmic specialization between adjoining Sertoli cells was observed as early as 20 days of age. In contrast, the specialization was not formed until 56 days of age in DES-treated rats. Furthermore, the delay in functional maturation of this structure as the blood-testis barrier was confirmed by intercellular tracer experiments. It is clear that neonatal administration of DES delayed the establishment of the blood-testis barrier for 4 weeks. Consequently, during this period, pachytene spermatocytes were exfoliated from the seminiferous epithelium without completion of meiosis.

A novel cell encapsulation method using photosensitive poly(allylamine alpha-cyanocinnamylideneacetate).

A photosensitive alpha-cyanocinnamylideneacetyl group was coupled to poly(allylamine) to obtain a photosensitive polymer. This photosensitive poly(allylamine alpha-cyanocinnamylideneacetate) can cross-link upon light exposure. Microcapsules were fabricated from alginate in contact with Ca+2 ion, followed by coating with the photosensitive poly(allylamine alpha-cyanocinnamylideneacetate). The microcapsules, thus formed, can be strengthened significantly by the light-induced cross-linking of poly(allylamine alpha-cyanocinnamylideneacetate). Only 16 capsules (out of 50) prepared from the photosensitive poly(allylamine alpha-cyanocinnamylideneacetate) fractured after 48 h of agitation. For microcapsules prepared from the unmodified poly(allylamine), 32 capsules fractured. The photo cross-linked capsular membrane was permeable to cytochrome C, moderately permeable to myoglobin, and least permeable to serum albumin. IW32 (a mouse leukaemia cell line) cells were entrapped and cultured within these microcapsules. The cells proliferated to a density of about 9 x 10(6) cells/ml in the capsules after 7 days of cultivation.

Bacterial adhesion at synthetic surfaces.

A systematic investigation into the effect of surface chemistry on bacterial adhesion was carried out. In particular, a number of physicochemical factors important in defining the surface at the molecular level were assessed for their effect on the adhesion of Listeria monocytogenes, Salmonella typhimurium, Staphylococcus aureus, and Escherichia coli. The primary experiments involved the grafting of groups varying in hydrophilicity, hydrophobicity, chain length, and chemical functionality onto glass substrates such that the surfaces were homogeneous and densely packed with functional groups. All of the surfaces were found to be chemically well defined, and their measured surface energies varied from 15 to 41 mJ. m(-2). Protein adsorption experiments were performed with (3)H-labelled bovine serum albumin and cytochrome c prior to bacterial attachment studies. Hydrophilic uncharged surfaces showed the greatest resistance to protein adsorption; however, our studies also showed that the effectiveness of poly(ethyleneoxide) (PEO) polymers was not simply a result of its hydrophilicity and molecular weight alone. The adsorption of the two proteins approximately correlated with short-term cell adhesion, and bacterial attachment for L. monocytogenes and E. coli also correlated with the chemistry of the underlying substrate. However, for S. aureus and S. typhimurium a different pattern of attachment occurred, suggesting a dissimilar mechanism of cell attachment, although high-molecular-weight PEO was still the least-cell-adsorbing surface. The implications of this for in vivo attachment of cells suggest that hydrophilic passivating groups may be the best method for preventing cell adsorption to synthetic substrates provided they can be grafted uniformly and in sufficient density at the surface.

Adsorption of low molecular weight proteins to hemodialysis membranes: experimental results and simulations.

Adsorption of alpha-lactalbumin and Cytochrome C on different hollow-fiber hemodialysis modules, whose main membrane constituent was polyacrylonitrile (PAN), polymethylmethacrylate (PMMA) or polysulfone (PS), were compared under different pH and flow rate conditions. These proteins were chosen as models of small scarce proteins like beta2-microglobulin from which the patient's blood should be epurated. Influence of pH suggests the importance of electrostatic interactions on a charged membrane since adsorption greatly decreases at pH 10.9 for Cytochrome C (pI = 10.6) and at pH 7.4 for alpha-lactalbumin (pI = 5). The difference in adsorbance between different membranes is most probably due to their different microstructures. However, the chemical nature of the support plays a non-negligible role since PMMA membranes have smaller initial adsorption rates than PAN membranes. This is correlated with the density of membrane surface charge showing the importance of electrostatic interactions. The influence of the wall shear rate on adsorption kinetics is analyzed through numerical simulations, in terms of transport limitation in the liquid phase and interfacial reaction.

In situ observation of bovine serum albumin-adsorbed stearic acid monolayer by Brewster angle microscopy.

The morphology of protein-adsorbed stearic acid monolayers containing a fluorescent probe (rhodamine B octadecylester perchloride: RBO) was observed by using fluorescence spectroscopy, fluorescence microscopy, and Brewster angle microscopy (BAM). The quenching of fluorescence of RBO was observed at the limiting area of the stearic acid monolayer. Thus, the fluorescence of RBO could be a good marker for packing of the matrix monolayer. On the other hand, the BAM image showed the morphology of not the matrix monolayer, but the adsorbed protein monolayer. Thus, the packing processes of the two could be distinguished. The present method is available for a protein that does not have visible absorption, such as bovine serum albumin (BSA). It is suggested that electrostatic interaction between matrix and protein molecules greatly affects the change in the morphology of the protein-adsorbed monolayer.

[Preparation of galactosyl-cytochrome C and studies on its hepatic targeting property].

This paper reports the preparation of the coupling compound between thiogalactose and cytochrome C(Cyt-C). The binding tests of this compound to asialoglycoprotein receptor have also been carried out in vivo. The results show that the pharmacokinetics of this compound is in accordance with the basic characteristic of receptor induced binding. The highest uptake of liver reaches to 32.9% and at the same time the conjugate remains 78.6% the biological activity of Cyt-C. It is possibly a hepatic targeting drug or a hepatic targeting carrier.

Permeability and charge-dependent adsorption properties of the S-layer lattice from Bacillus coagulans E38-66.

We investigated the permeability properties of the oblique S-layer lattice from Bacillus coagulans E38-66 after depositing cell wall fragments on a microfiltration membrane, cross-linking the S-layer protein with glutaraldehyde, and degrading the peptidoglycan with lysozyme. Comparative permeability studies on such multilayered S-layer membranes and suspended S-layer vesicles from thermophilic members of the family Bacillaceae with use of the space technique (M. Sára and U. B. Sleytr, J. Bacteriol. 169:4092-4098, 1987) revealed identical molecular exclusion limits (M. Sára and U. B. Sleytr, J. Membr. Sci. 33:27-49, 1987). Examination of the S-layer lattice from B. coagulans E38-66 with the S-layer membrane technique revealed unhindered passage for molecules up to the size of myoglobin (M(r) 17,000). The molecular dimensions of this protein (2.8 by 3.2 by 4.5 nm) correspond approximately to the size of the ovoid-shaped pore previously shown by high-resolution electron microscopy of negatively stained S-layer self-assembly products (D. Pum, M. Sára, and U. B. Sleytr, J. Bacteriol. 171:5296-5303, 1989). Chemical modification of the S-layer protein and comparative labeling, adsorption, and permeability studies clearly demonstrated that (i) in the native state, free amino and carboxyl groups are present on the outer S-layer face and in the interior of the pores and (ii) electrostatic interactions between these groups prevent unspecific adsorption of the S-layer in vivo.

Synthesis of a cytochrome c derivative with prolonged in vivo half-life and determination of ascorbyl radicals in the circulation of the rat.

Since cytochrome c and acetylated cytochrome c disappear from the circulation with a half-life of 4 min, these proteins cannot be used for in vivo detection of superoxide radicals and related metabolites. To determine superoxide and other radicals in vivo, a cytochrome c derivative (SMAC) was synthesized by linking 1 mol of poly(styrene-co-maleic acid) butyl ester (SM) to cytochrome c, followed by acetylation of its lysyl amino groups. SMAC retained 8 and 80% of cytochrome c activity to react with ascorbyl and superoxide radicals, respectively. However, SMAC did not serve as a substrate for cytochrome c reductase and cytochrome c oxidase. When injected intravenously to the rat, SMAC circulated bound to albumin with a half-life of 130 min. SMAC was rapidly reduced in the circulation of intact animals. Treatment of animals with paraquat markedly enhanced the reduction of the circulating SMAC. We have synthesized an SM-conjugated superoxide dismutase (SOD) derivative (SM-SOD) that circulates bound to albumin with a half-life of 6 h. Kinetic analysis revealed that SM-SOD effectively inhibited the superoxide-dependent reduction of SMAC either in the presence or absence of 0.5 mM albumin. However, the reduction of the circulating SMAC was not inhibited by SM-SOD both in normal and paraquat-treated animals. Plasma samples from both animal groups also reduced cytochrome c and SMAC by an SOD-insensitive mechanism. However, after treatment with ascorbate oxidase, both plasma samples lost their activity to reduce cytochrome c and SMAC. These and other results suggest that ascorbyl radical might principally be responsible for the reduction of circulating SMAC and that plasma levels of ascorbyl radical might increase in paraquat-treated animals.

A new method of determining the solute permeability of hollow-fiber dialysis membranes by means of laser lights traveling along optic fibers.

To develop a new method of determining solute permeability more simply and accurately, the authors employed light from a laser traveling along quartz optic fibers. Dialysis experiments at 310 K were made with a single hollow fiber containing aqueous test solutes. A membrane tube was sealed at either end with quartz optic fibers. Helium-neon and helium-cadmium laser lights emitted from one of these optic fibers into the test solution at wavelengths of 543 and 442 nm for vitamin B12 and cytochrome-C, respectively, were caught by the other optic fiber and detected with a silicon photodiode. The solute permeability for cytochrome-C obtained by this method was almost in agreement with that for beta-2-microglobulin by the radioisotope method. This study demonstrates the usefulness of light from a laser traveling along quartz optic fibers in determining the solute permeability of hollow-fiber dialysis membranes.