Sterilization using pulsed white light.

Pulsed light is a non-thermal sterilization method that uses brief intense pulses or flashes of white light to kill micro-organisms. This article discusses tests performed on blow/fill/seal containers. The results suggest that pulsed light has potential as a terminal sterilization method for filled and sealed transmissive products and packages. It also examines the effectiveness of pulsed light in eliminating water-borne pathogenic organisms.

Beta-amyloid induces apoptosis in human-derived neurotypic SH-SY5Y cells.

Alzheimer's disease is primarily characterized by neurofibrillary tangles, senile plaques, and neurodegeneration. The major component of senile plaques is the beta-amyloid peptide (beta A4), which has been shown to be toxic to neurons in vitro. To date, the mechanism of beta A4-induced neurotoxicity has not been determined in human-derived neurons. In this report, we present evidence that programmed cell death, or apoptosis, is involved in the neurotoxic activity of beta A41-40 and beta A425-35 in the human-derived neurotypic cell line SH-SY5Y cells. The evidence for beta A4-induced apoptosis includes: (1) labeling of cell nuclei for DNA nicked ends; (2) morphological changes in ultrastructure that are consistent with apoptosis (shrunken cells with pyknotic nuclei); (3) DNA laddering which can be blocked by aurintricarboxylic acid (ATA), an inhibitor of apoptosis; and (4) marginal release of intracellular lactate dehydrogenase (LDH), an indicator of necrosis. These results suggest that apoptosis is the major event involved in beta A4-induced cytotoxicity in SH-SY5Y cells. A variety of reagents were tested to determine their activities against beta A4-induced DNA laddering. Nerve growth factor and free radical scavengers were inactive in this system. While ATA blocked DNA laddering resulting from either beta A4 or okadaic acid treatment, Congo red specifically attenuated only beta A4-induced DNA fragmentation. These results suggest that compounds which bind fibrillar beta-peptides can protect this human neurotypic cell line against apoptosis induced by beta A4.

Development and characterization of a monoclonal antibody 369.2B specific for the carboxyl-terminus of the beta A4 peptide.

The beta-amyloid deposits in Alzheimer's diseased (AD) brain are made up mainly of beta A4 peptides which show both N- and C-terminal heterogeneity. The predominant C-terminal species, identified by peptide sequencing of purified beta-amyloid, end either at position 40 or 42 of the beta A4 peptide. The distribution of these two beta A4 species in postmortem tissue as well as their generation in vitro could not be addressed previously due to the lack of specific antibodies that could differentiate them. This report describes the generation of a highly specific monoclonal antibody, MAb 369.2B which was raised against a synthetic peptide consisting of the C-terminus of the 1-42 beta A4 species. MAb 369.2B does not recognize the shorter beta A4 species 1-40 in solution or in solid phase. Furthermore, both beta A4 1-40 and 1-43 were unable to absorb out the antibody when used immunocytochemically. The regional distribution of MAb 369.2B immunoreactivity in AD and non-AD brain samples were compared to MAb 286.8A, an antibody raised against the N-terminal region of beta A4. Overall, the staining patterns were very similar. In AD cases with extreme vascular involvement, the N-terminal (MAb 286.8A) antibody was more likely to label the vascular basement membrane of affected vessels, and to label them more uniformly. In addition, preliminary quantitative analyses revealed that the C-terminal antibody labeled fewer, larger plaques than the N-terminal antibody. Qualitative analyses revealed that these smaller plaques were typically subregions of the larger plaques. The data corroborate the biochemical findings of N-terminal raggedness in beta A4 plaques. Further studies are required to explain this differential pattern of deposition.

Postsynaptic gephyrin immunoreactivity exhibits a nearly one-to-one correspondence with gamma-aminobutyric acid-like immunogold-labeled synaptic inputs to sympathetic preganglionic neurons.

Peripheral regulation of cardiovascular function is fundamentally influenced by central excitation and inhibition of sympathetic preganglionic neurons in thoracic spinal cord. This electron microscopy study investigated whether the gamma-aminobutyric acid (GABA)-ergic and glycinergic inhibitory innervation of sympathetic preganglionic neurons arises from mutually exclusive afferent populations. Sympathetic preganglionic neurons were retrogradely labeled with cholera beta subunit. GABAergic terminals were identified using strict quantitative statistical analyses as those boutons containing significantly elevated levels of GABA-like immunogold labeling (GABA+). Glycinergic terminals were classified as those boutons opposite postsynaptic gephyrin immunostaining containing background levels of GABA-like immunogold labeling (gephyrin+/GABA- association). Approximately 43% of the synaptic terminals that contacted sympathetic preganglionic somata and proximal dendrites and that were opposite gephyrin were GABA-; the remaining 57% were GABA+. Only two GABA+ boutons (4%) that synapsed on identified sympathetic preganglionic neuron (SPN) processes were not opposite gephyrin immunostaining (GABA+/gephyrin- association). GABA-/gephyrin+ associations were anticipated given prior anatomical, physiological, and pharmacological data. The observed nearly one-to-one correspondence between postsynaptic gephyrin immunoreactivity and GABA+ boutons was unexpected. Prior physiological and pharmacological experiments suggest that the postsynaptic effects of GABAergic inputs to sympathetic preganglionic neurons are mediated by activation of GABAA receptors. Those data, the present results, and other molecular, biochemical, and anatomical studies of gephyrin in the central nervous system (CNS) are consistent with two hypotheses: 1) Postsynaptic gephyrin is associated with GABAA receptors in the membranes of sympathetic preganglionic neurons, and 2) GABA+/gephyrin+ associations do not necessarily predict colocalization of GABA and glycine within single boutons synapsing on sympathetic preganglionic somata and dendrites.

Glycine-like immunoreactive input to sympathetic preganglionic neurons.

The organization of glycine-like immunoreactive (GLY-LIR) processes was investigated within the sympathetic preganglionic neuropils of male Sprague-Dawley rats and pigeons (Columba livia). Sympathetic preganglionic neurons were retrogradely labeled with horseradish peroxidase following injections into the superior cervical ganglion in rats or into the avian homologue of the mammalian stellate ganglion (paravertebral ganglion 14) in pigeons. Glycine-like immunoreactivity was visualized using postembedding immunoperoxidase and immunogold labeling methods. The neuropils surrounding pigeon sympathetic preganglionic neurons in the principal preganglionic cell column (nucleus of Terni) and in the nucleus intercalatus contained numerous GLY-LIR puncta. Many of these processes appeared to be 'terminal-like' swellings which closely apposed retrogradely labeled preganglionic perikarya and proximal dendrites. GLY-LIR somal and dendritic processes were intermingled among retrogradely labeled preganglionic neurons in the nucleus of Terni. None of these GLY-LIR cells were retrogradely labeled. The neuropils surrounding sympathetic preganglionic neurons in the rat also contained numerous GLY-LIR puncta; there were, however, qualitative differences in the density of such profiles across the preganglionic subnuclei. Within the central autonomic and intercalated regions there were numerous GLY-LIR processes, many of which closely apposed retrogradely labeled sympathetic preganglionic somas and proximal dendrites. Within the principal preganglionic cell column, the nucleus intermediolateralis pars principalis (Ilp), there were very few GLY-LIR 'terminal-like' swellings closely apposed to cell bodies in regions of high somal packing density. In regions were this density diminished, GLY-LIR puncta closely apposed retrogradely labeled perikarya and proximal dendritic processes. GLY-LIR spinal neurons were never observed to be within Ilp proper but were present in areas immediately dorsal (lateral lamina V), medial and ventral (lateral lamina VII). GLY-LIR neurons were never retrogradely labeled. The ultrastructural features of GLY-LIR terminals within the sympathetic preganglionic neuropils of both vertebrates were nearly identical. GLY-LIR terminal boutons formed synaptic contacts with retrogradely labeled preganglionic somas as well as with large and medium-sized proximal dendrites. The majority of identified GLY-LIR terminals, however, contacted non-retrogradely labeled medium and small caliber dendrites within the preganglionic neuropils. Ninety-eight percent of GLY-LIR synapses formed symmetric specializations with the postsynaptic element. Ninety-six percent of the GLY-LIR terminal boutons contained some combination of pleomorphic vesicles. These light and electron microscopic observations support the hypothesis that glycine is localized in terminals presynaptic to sympathetic preganglionic perikarya and dendrites.(ABSTRACT TRUNCATED AT 400 WORDS)

Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules.

Pinocytosis of the fluorescent dye lucifer yellow labels elongated, membrane-bound tubular organelles in several cell types, including cultured human monocytes, thioglycolate-elicited mouse peritoneal macrophages, and the macrophage-like cell line J774.2. These tubular structures can be identified as lysosomes by acid phosphatase histochemistry and immunofluorescence localization of cathepsin L. The abundance of tubular lysosomes is markedly increased by treatment with phorbol 12-myristate 13-acetate (10 ng/ml). When labeled by pinocytosis of microperoxidase and examined by electron microscopic histochemistry, the tubular lysosomes have an outside diameter of approximately 75 nm and a length of several micrometers; they radiate from the cell's centrosphere in alignment with cytoplasmic microtubules and intermediate filaments. Incubation of phorbol myristate acetate-treated macrophages at 4 degrees C or in medium containing 5 microM colchicine or nocodazole at 37 degrees C leads to disassembly of microtubules and fragmentation of the tubular lysosomes. Return of the cultures to 37 degrees C or removal of nocodazole from the medium leads to reassembly of microtubules and the reappearance of tubular lysosomes within 10-20 min. We conclude that microtubules are essential for the maintenance of tubular lysosome morphology and that, in macrophages, a significant proportion of the lysosomal compartment is contained within these tubular structures.

Effect of alterations in the size of the vacuolar compartment on pinocytosis in J774.2 macrophages.

J774.2 macrophages cultured in medium containing 10 mg/ml sucrose accumulate the sugar by pinocytosis and become highly vacuolated, due to the sugar's osmotic effect within the vacuolar compartment. When such cells are incubated in medium containing 0.5 mg/ml invertase, the enzyme reaches the sucrose vacuoles by pinocytosis, then cleaves the sugar to more permeant monosaccharides. Within 4 hours, the vacuoles shrink to smaller, phase-dense organelles (Cohn and Ehrenreich, 1969, J. Exp. Med., 129:201). We have used this reversible expansion of the lysosomal compartment to address two questions: (1) Does the increased size of the lysosomal compartment affect pinocytic accumulation of solute, and (2) what is the fate of the vacuolar membrane and its soluble content during invertase-induced vacuole shrinkage? Using lucifer yellow (LY) as a probe for pinocytic fluid influx and efflux, we found that vacuolated cells accumulated 30-50% less LY than controls and returned to higher rates of pinocytosis after invertase-induced vacuole shrinkage. A similar reduction in LY accumulation was achieved after feeding cells latex beads to increase the size of the lysosomal compartment. Thus, treatments that increased the size of the lysosomal compartment reduced solute accumulation via pinocytosis. A dramatic shrinkage of LY-containing sucrose vacuoles followed pinocytosis of invertase. Despite this reduction in size of the LY-containing vacuoles, the overall rate of LY efflux did not increase significantly during invertase-induced vacuole collapse. Electron microscopy revealed that during shrinkage, the excess vacuolar membrane was compressed into whorled membranous organelles (residual bodies), with fluid markers (colloidal gold and, by inference, LY) trapped inside. The trapping of LY inside lysosomes as J774.2 macrophages returned to their normal dimensions indicates that nearly all of the surplus membrane contents were removed from circulation as well.

Observations on the vimentin-10-NM filaments during mitosis in BHK21 cells.

Mitotic BHK21 cells were examined by immunofluorescence optical sectioning after staining with anti-vimentin. Throughout all stages of mitosis, intact 10-nm filaments were observed to form a cage around the mitotic spindle. These observations were confirmed by serial section electron microscopy which demonstrated assembled 10-nm filaments in the cytoplasm surrounding the spindle. Therefore, in contrast to previous reports, mitotic BHK21 cells show a similar cage-like 10-nm filament arrangement, as found in a variety of other cell types.

Studies on intercellular LETS glycoprotein matrices.

Intercellular matrices secreted by chick embryo fibroblasts in culture were studied by scanning electron microscopy. Cell-cell contact is a prerequisite for the expression of such matrices. The smallest fiber detected by transmission electron microscopy is 5--10 nm in diameter. These matrix fibers tend to cluster to form bundles. Immunofluorescence and immunoferritin procedures reveal that LETS protein is one of the components of the matrices. The matrices are isolated from other cellular organelles by detergent treatment. More than 90% of the proteins in cell-free matrices are LETS protein, suggesting that the matrices are probably made of only one component--LETS protein. Since the solubilization of matrices requires beta-mercaptoethanol, LETS protein matrices may be the first known polymer system in nature to use disulfide linkage as an intermolecular polymerization vehicle. Collagen does not appear to be involved in such matrices. The LETS protein matrix supports the morphological conversion of rounded cells into spindle-shaped, and also promotes myoblast fusion. It does not, however, exert an effect upon cell growth, the rate of glucose uptake or protease production.

Characterization of the intermediate (10 nm) filaments of cultured cells using an autoimmune rabbit antiserum.

An antiserum has been found in a nonimmunized rabbit which reacts strongly with a system of filaments in various fibroblasts, epithelial cells, macrophages and neuroblastoma. These filaments are distinct from the actin microfilament bundles visualized by an antibody against actin, and they are not affected by brief treatment with cytochalasin B. The pattern of these filaments somewhat resembles that described for microtubules, but the filaments could be clearly distinguished from microtubules by a comparison of their respective immunofluorescent patterns during cell division. In response to the drugs colcemid and vinblastine, the filaments reacting with this preimmune serum condense to form a compact perinuclear coil of fibers, a distribution and behavior in agreement with that previously described for the 10 nm or intermediate filaments studied by electron microscopy. Further evidence supporting our conclusion that this antiserum reacts with intermediate filaments is provided by a comparison of electron micrographs and the immunofluorescent patterns from parallel cell cultures. To identify the antigens reacting with this antiserum we have used the new technique of immuno-autoradiography on SDS gels of whole cell extracts. Two reactive polypeptide chains have been identified with apparent molecular weights of 56,000 and 30,000 daltons.