Survival Rate and Enzyme Activities ofLactobacillus acidophilusFollowing Frozen Storage

The ability of two strains of Lactobacillus acidophilus, CRL 640 and CRL 800, to survive and retain their biological activities under frozen storage was determined. Freezing and thawing, as well as frozen storage, damaged the cell membrane, rendering the microorganisms sensitive to sodium chloride and bile salts. Both lactic acid production and proteolytic activity were depressed after 21 days at -20 degreesC, whereas beta-galactosidase activity per cell unit was increased. Cell injury was partially overcome after repair in a salt-rich medium. Copyright 1998 Academic Press.     

Conditionally adherent growth of serum-independent CHO cells for automated drug screening and biopharmaceutical production.

SSF3 is a CHO cell line adapted for growth in protein-free medium. It grows in suspension unless serum-derived attachment factors such as vitronectin are added to the medium. Serum-independent cell lines, which adhere to the substrate after induction with dexamethasone or constitutively, were created by transfection with a human vitronectin gene under control of the mouse mammary tumor-virus promoter. Substrate attachment and SSF3VN-cell spreading could be prevented with an RGD peptide (arginine-glycine-aspartic acid) confirming that attachment is mediated by an intregrin receptor. Hormone-inducible attachment could be blocked by glucocorticoid antagonist promegestone. All steps in the isolation of stable transfected SSF3VN cell lines could be done in a chemically defined medium avoiding the risk of introduction of serum-derived infectious agents. SSF3VN cells could be grown in protein-free medium in solid-phase large-scale bioreactors. Application in microplates as used in high-throughput screening was demonstrated in an assay of Ca(2+) release from internal stores induced by agonist-binding to recombinant human metabotropic glutamate receptor hmGluR1b.     

Giardia and Cryptosporidium in inflowing water and harvested shellfish in a lagoon in Southern Italy

Giardia and Cryptosporidium spp. are important enteric protozoan pathogens for humans and animals, and have been found to contaminate water as well as edible shellfish all over the world. This is the first study to simultaneously investigate the presence of Giardia and Cryptosporidium in inflowing water and harvested shellfish in a geographically closed environment (Varano Lagoon, Southern Italy). Samples of treated wastewater were collected each month - at the outlet from the treatment plant, and downstream at the inlet into the lagoon - from the channels flowing into the Lagoon, together with specimens of Ruditapes decussatus and Mytilus galloprovincialis from shellfish-farms on the same lagoon. Giardia cysts were found by immunofluorescence (IF) microscopy in 16 out of 21 samples of treated wastewater and in 7 out of 21 samples from downstream water channels, and viable cysts were also detected by a beta-giardin RT-PCR. G. duodenalis Assemblages A and B were identified by small ribosomal subunit (18S-rDNA) and triosephosphate isomerase (tpi)-PCR, followed by sequencing. Cryptosporidium oocysts were found by IF in 5 out of 21 wastewater samples, and in 8 out of 21 samples from water channels. Molecular analysis identified the zoonotic species Cryptosporidium parvum by oocyst wall protein (COWP)-PCR and sequencing. Higher concentrations of Giardia cysts than Cryptosporidium oocysts were registered in almost all wastewater and water samples. IF and molecular testing of shellfish gave negative results for both protozoa. Wastewaters carrying Giardia and Cryptosporidium (oo)cysts are discharged into the Lagoon; however, the shellfish harvested in the same environment were found to be unaffected, thus suggesting that physical, ecological and climatic conditions may prevent contamination of harvested shellfish.     

Polymorphism of myosin among skeletal muscle fiber types

An immunocytochemical approach was used to localize myosin with respect to individual fibers in rat skeletal muscle. Transverse cryostat sections of rat diaphragm, a fast-twitch muscle, were exposed to fluorescein-labeled immunoglobulin against purified chicken pectoralis myosin. Fluorescence microscopy revealed a differential response among fiber types, identified on the basis of mitochondrial content. All white and intermediate fiber but only about half of the red fiber reacted with his antimyosin. In addition, an alkali-stable ATPase had the same pattern of distribution among fibers, which is consistent with the existence of two categories of red fibers. The positive response of certain red fibers indicates either that their myosin has antigenic determinants in common with "white" myosin, or that the immunogen contained a "red" myosin. Myosin, extracted from a small region of the pectorlis which consists entirely of white fibers, was used to prepare an immunoadsorbent column to isolate antibodies specific for white myosin. This purified anti-white myosin reacted with the same fibers of the rat diaphragm that had reacted with the white, intermediate, and some red fibers are sufficiently homologous to share antigenic determinants. In a slow-twitch muscle, the soleus, only a minority of the fiber reacted with antipectoralis myosin. The majority failed to respond; hence, they are not equivalent to intermediate fibers of the diaphragm; despite their intermediate mitochondrial content. Immunocytochemical analysis of two different musles of the rat has demonstrated that more than one isoenzyme of myosin can exist in a single muscle, and that individual fiber types can be recognized by immunological differences in their myosin. We conclude that, in the rat diaphragm, there are at least two immunochemically distinct types of myosin and four types of muscle fibers: white, intermediate, and two red. We suggest that these fibers correspond to the four types of motor units described by Burke et al. (Burke, R. E., D. N. Levine, P. Tsairis, and F. E. Zajac, III 1973. J. Physiol. (Lond) 234:723-748.)in the cat gastrocnemius.`