Stabile production of a thrombin resistant pro-urokinase derivative (PRO-UKS1) by Namalwa KJM-1 cells adapted to serum-free medium

Pro-UKS1 was designed as a thrombin-resistant derivative of pro-urokinase (pro-UK) by introducing a glycosylation site using site-directed mutagenesis. An expression plasmid for pro-UKS1, pMo1UKS1SEd1-5, was constructed and introduced into Namalwa KJM-1 cells (Hosoiet al., 1988), and cells resistant to G418 and Methotrexate (MTX) were obtained. Amongst them, the highest pro-UKS1 producer (resistant to 500 nM of MTX), clone 41-8, was selected and further characterized. Clone 41-8 was cultured in serum-free ITPSGF medium (Hosoiet al., 1988). Under the conventional conditions, the concentration of pro-UKS1 reached 26 g ml^–1. Addition of glucose and tri-iodothyronine (T₃) improved productivity, and the maximal productivity of pro-UKS1 was 67 g ml^–1 day^–1. In this conditioned medium, content of pro-UKS1 was above 80% of total proteins.Abbreviations BSA bovine serum albumin - dhfr dihydrofolate reductase - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - kb kilobase pairs - kDa kilodaltons - MTX Methotrexate - PBS phosphate buffered saline - pro-UK pro-urokinase - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - T₃ tri-iodothyronine - Tween-PBS phosphate buffered saline containing 0.05% Tween 80     

Parallel bioassay of litorin and phyllolitorins on smooth muscle preparations

Phyllolitorin and Leu8-phyllolitorin, two nonapeptide amides from the skin of the South American frog Phyllomedusa sauvagei, are representatives of a novel bombesin subfamily, characterized by the occurrence in their molecule of a serine residue substituting the usual histidine residue at position 3 from the C-terminus. In parallel bioassay on ten different smooth muscle preparations and rat blood pressure, phyllolitorin and Leu8-phyllolitorin were virtually equiactive, but the two peptides appeared remarkably less potent that litorin in all test preparations, except the rat urinary bladder. The shape of contractions produced by the phyllolitorins and promptness of cessation of their action upon washing seem to indicate a looser binding of these peptides to their receptors and/or a more rapid inactivation, in comparison to litorin.     

Binding of fluorescent lectins to the surface of germ cells from fetal and early postnatal mouse gonads

Fluorescent lectins were used to study the chemical nature of carbohydrate moieties present on the surface of female and male germ cells isolated from mouse gonads during fetal and early posnatal development. Concanavalin A (ConA), lens culinaris agglutinin (LCA), ricinus communis agglutinin (RCA_I) and wheat germ agglutinin (WGA) bound intensely to the germ cell plasma membrane at all stages studied. Other lectins such as ulex europaeus agglutinin (UEA_I) and agglutinin (SBA) did not bind or bound moderately (SBA to female germ cells only). Distinct developmental-related changes were observed when female germ cells were labeled with fluorescein-conjugated peanut agglutinin (PNA) or dolichos biflorus agglutinin (DBA). DBA and PNA binding was absent or weak in fetal female and male germ cells, but became intensely positive in oocytes in the immediate postnatal period. The percentage of oocytes stained with DBA increased during the first three days after birth, and from day 3–4 onwards all oocytes were strongly labeled. I suggest that these changes in lectin binding reflect changes in biochemical structure of the oocyte surface related to differentiative events occurring in the mouse ovary immediately after birth.     

Taenia taeniaeformis: characterization of larval metabolic products and growth of host gastric cells in vitro

Development of larvae of the cestode parasite Taenia taeniaeformis in the liver of rats induces gross hyperplasia of the gastric mucosa and excessive mucus production in the stomach without any direct contact with the stomach. Because the taeniid larvae are known to elaborate excretory-secretory (E-S) product in vivo and in vitro, the product was analyzed further, and its effects on cultured rat and dog stomach cells were investigated. In vitro E-S product contained less negatively charged glycosaminoglycan than either heparin or chondroitin sulfate, and proteins of various molecular weights. It stimulated the growth of both rat and dog stomach cells at concentrations of 3-9 micrograms protein/ml culture medium. At a concentration of 30 micrograms protein/ml culture medium, it stimulated hexosamine production in the cells up to 20 times, and multiple intracytoplasmic granules were found in both rat and dog cultured cells by light and electron microscopy. These results suggest that larval E-S product may be involved in the induction of gastric hyperplasia and hypermucus secretion.     

Extracellular glycosaminoglycans (GAG) released by chick embryonic fibroblasts

Summary Administration of Concanavalin A (Con A) to cultured skin fibroblasts derived from chick embryos at two developmental stages produce variations in the relative concentration of individual glycosaminoglycan (GAG) secreted by the cells. This effect is different: at 7 days (increase of hyaluronic acid and dermatan sulphate and decrease of chondroitin sulphate) and at 14 days (dermatan sulphate is not detectable).All the cells bind the Con A specifically, but a different pattern of agglutination is present in fibroblasts of the two embryonic ages. Since Con A is well known to bind carbohydrate-containing surface proteins, the result suggests that the release of GAG by chick embryonic fibroblasts can be modulated by cell surface receptors.     

The paramedial neurosecretory cells of the suboesophageal ganglion of the cricket,Teleogryllus commodus (Walk.)

Summary Ovariectomy, performed immediately after the final hatch, caused a reduction of stainable (neurosecretory?) material in the paramedial neurosecretory cells (PNC) (A-type) of the suboesophageal ganglion in 10 day-old females of Teleogryllus commodus (Walk.). A concomitant increase in nuclear volume and in the incorporation of ³⁵S-cysteine indicates increased synthesis of neurosecretory material. From these findings it is concluded that more stainable material is secreted in the cerebral neurohaemal organ after Ovariectomy. A functional relationship between the PNC and the ovaries is suggested.     

Isolation and identification of epithelial-like cells in culture by a collagenase-separation technique

Summary An operational criterion for the identification and isolation of epithelial-like (E) cells, based on their ability to cover and protect, a collagen gel from the action of collagenase, has been developed. The E cells isolated by this collagenase-separation technique (CST) exhibited the ultrastructural features, including desmosomes and abundant tonofilaments, that are considered characteristic of this cell type. Unlike confluent cultures of fibroblast-like (F) cells, E cells were not found to have large external transformation-sensitive (LETS) protein on their surface membranes. The CST provides a nondestructive, and efficient means of identifying and isolating E cells from mixed populations.     

The goblet cavity matrix in the larval midgut of Hyalophora cecropia

The larval midgut of the silkmoth Hyalophora cecropia was examined using scanning electron microscopy. Goblet cells were observed to contain within their cavities a matrix plug. This matrix material was extruded onto the lumen side of the epithelium when the tissue was stretched. The rôle of this matrix material in maintenance of the capacity of the midgut to transport ions in vivo and in vitro is discussed.     

Growth of endothelial and HeLa cells on a new multipurpose microcarrier that is positive,negative or collagen coated

A new cell culture microcarrier that can be covalently bonded by cell attachment proteins and can be thin-sectioned for electron microscopy was synthesized. It was easily made by sulfonating cross-linked polystyrene beads for a negative surface charge followed by covalent attachment of polyethylenimine for a positive charge. Cell attachment proteins, e.g. collagen, was covalently bonded directly to the microcarrier using a carbodiimide or after activating the microcarrier surface with glutaraldehyde. HeLa-S3 cells attached, spread and grew to confluence more efficiently on the positive microcarriers and those coated with collagen than on the negative ones. Endothelial cells grew best on those with a negative surface charge. The nature of the microcarrier surface was not the only aspect involved in cell adhesion but also the type of serum proteins adsorbed. Qualitatively different proteins coated the microcarriers depending upon whether the carrier was negative, positive or coated with collagen. Comparison of various types of available microcarriers indicated that the modified cross-linked polystyrene beads used here were best for transmission and scanning electron microscopy. Endothelial cells grown on the microcarriers had the same ultrastructure as cells grown in monolayers in culture dishes. Of a variety of microcarriers tested the modified cross-linked polystyrene beads were the only ones that could be used for both ultrastructural and biochemical techniques.     

Ultrastructure of the sporophyte foot-gametophyte vaginula complex inTimmiella barbuloides (Brid.) Moenk

The sporophyte foot of the mossTimmiella barbuloides consists of an unistratose epidermis of transfer cells, a parenchymatous cortex, and a small central strand consisting only of hydroids. The parenchymatous tissue of the vaginula develops one layer of transfer cells opposite the foot, whose lower extremity extends into the gametophyte stem's central strand. From the bottom to the top of the foot the ultrastructure of the sporophyte transfer cells shows some gradual changes that appear related to a functional specialization of these cells. According to a centripetal gradient, the quantity of plastid starch progressively lessens in both vaginula parenchyma and foot cortex. the observed morphological patterns suggest that in the foot-vaginula complex nutrients are translocated radially up to the sporophyte central strand.