Secretion of Recombinant Pediocin PA-1 by Bifidobacterium longum, Using the Signal Sequence for Bifidobacterial α-Amylase

A recombinant DNA, encoding the chimeric protein of the signal sequence for bifidobacterial α-amylase mature pediocin PA-1, was introduced into Bifidobacterium longum MG1. Biologically active pediocin PA-1 was successfully secreted from the strain and showed bactericidal activity against Listeria monocytogenes and the same molecular mass as native pediocin PA-1.     

Cancer Cell Analyses at the Single Cell-Level Using Electroactive Microwell Array Device

Circulating tumor cells (CTCs), shed from primary tumors and disseminated into peripheral blood, are playing a major role in metastasis. Even after isolation of CTCs from blood, the target cells are mixed with a population of other cell types. Here, we propose a new method for analyses of cell mixture at the single-cell level using a microfluidic device that contains arrayed electroactive microwells. Dielectrophoretic (DEP) force, induced by the electrodes patterned on the bottom surface of the microwells, allows efficient trapping and stable positioning of single cells for high-throughput biochemical analyses. We demonstrated that various on-chip analyses including immunostaining, viability/apoptosis assay and fluorescent in situ hybridization (FISH) at the single-cell level could be conducted just by applying specific reagents for each assay. Our simple method should greatly help discrimination and analysis of rare cancer cells among a population of blood cells.     

Continuous culture of a recombinantEscherichia coli and plasmid maintenance for tryptophan production

Summary Production of tryptophan by a temperature sensitive recombinant microorganism (Escherichia coli W3110 trpLDtrpR ^ts tna ^– (pCRT185)) was investigated. In a single-stage continous culture, at an elevated temperature, 42°C (derepressed condition), tryptophan concentration increased in an early phase of the fermentation, and then gradually decreased with time. The reduction in the production rate was mostly due to the segregation of the plasmid and subsequent increase of plasmid-free cells. However, the plasmid could be maintained stable at 37°C, with repressed condition oftrp-operon, over 200 generations. A two-stage continuous culture system, i.e. cell growth was maintained in the first stage at 37°C and gene expression was induced in the second stage at 42°C, was therefore tested to improve the performance of the fermentation system. Operation of the two-stage system showed that the plasmid stability was significantly improved, and the specific rate of tryptophan production was maintained almost constant for more than 500 hours in the second stage.     

Resistance to the cytolytic action of lymphotoxin and tumor necrosis factor coincides with the presence of gap junctions uniting target cells

The resistance of target cells to the cytolytic action of lymphotoxin (LT) and recombinant tumor necrosis factor (rTNF) has been investigated by using clonally derived cell lines with defined gap junction-mediated, intercellular communication properties. Gap junction-competent Chinese hamster ovary cells are normally insensitive to the action of LT/TNF. However, treatment with 12-o-tetradecanoylphorbol-13-acetate, which promotes the loss of gap junctions, or culturing at low cell density to reduce intercellular contacts, significantly increased their sensitivity to LT/TNF. The LT/TNF-sensitive murine CL-1D and L929 cell lines, which in normal culture conditions are unable to form gap junctions, were not changed in their susceptibility to LT/TNF after treatment with phorbol ester or low culture density. However, the formation of gap junctions by CL-1D can be promoted by treatment with 8-bromo-cyclic adenosine monophosphate (1 mM), and this treatment completely suppressed the ability of LT and rTNF to kill CL-1D. Additionally, the LA25-normal rat kidney cell line, which is infected with a temperature-sensitive mutant of Rous sarcoma virus (LA25), is gap junction-competent and resistant to the effects of LT at the restrictive temperature (39 degrees C). However, when shifted to the permissive temperature (33 degrees C), LA25-normal rat kidney cells express the pp60v-src viral gene product, lose their ability to form gap junctions, and become sensitive to the lytic activity of LT. The results demonstrate that the expression of the retroviral pp60v-src, a tyrosine protein kinase, is sufficient to render cells susceptible to the lytic effects of LT and rTNF. Collectively, these experiments demonstrate a strong correlation between the resistance of target cells to the action of LT/TNF and their ability to cooperate metabolically through gap junctions. The results do not completely exclude the possibility that other mechanisms, such as LT receptor modulation, are also occurring under these experimental conditions. These data also suggest that a possible physiologic function of the stable cytotoxic lymphokines is to induce cytolysis/cytostasis of cells that have lost gap junctional contact, such as those in the process of mitosis or metastasis that have separated from the main tissue mass.     

Genotype of Yupik Eskimos with congenital adrenal hyperplasia due to 21-hydroxylase deficiency

Summary An A-to-G transition in the second intron was the sole mutation detected in four Yupik Eskimo patients with salt-wasting congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency. Allele-specific hybridization should be an efficient means of performing prenatal diagnosis of the disease in this highly inbred population.     

Studies of myofibrillar ATPase in ragweed-sensitized canine pulmonary smooth muscle

The maximal shortening velocities of tracheal and pulmonary vascular smooth muscle from ragweed-sensitized dogs were significantly higher than those of muscles from their littermate controls. Myofibrils of tracheal and pulmonary vascular smooth muscle from ragweed-sensitized and control dogs were obtained with use of Triton X-100 homogenizing solution. The myofibrillar adenosinetriphosphatase (ATPase) activities of the sensitized tissues were significantly higher (P less than 0.05) than those of their respective controls.     

Properties of a prolactin receptor from the rabbit mammary gland

Receptors for human, simian, ovine, bovine and murine prolactin, human growth hormone and human placental lactogen have been identified in plasma-membrane-containing subcellular particles isolated from rabbit mammary glands. The association and dissociation of (125)I-labelled prolactin are time- and temperature-dependent processes, both being maximal at 37 degrees C. (125)I-labelled prolactin prepared by the enzymic iodination procedure with lactoperoxidase binds better to receptors than does the preparation obtained by using chloramine-t as the oxidizing agent. The binding of (125)I-labelled prolactin to receptors is strongly influenced by pH and ionic composition but not by many low-molecular-weight compounds tested, e.g. steroids, nucleotides and several drugs. Receptor activity is sensitive to trypsin and phospholipase C digestion, suggesting that protein and phospholipid moieties are essential for the binding of (125)I-labelled prolactin. The binding of (125)I-labelled prolactin to receptors is a saturable and reversible process. Scatchard and Lineweaver-Burk analyses suggest that (125)I-labelled prolactin has a high affinity for its receptor. Binding of (125)I-labelled prolactin to receptors does not result in the destruction of the hormone. Considerable prolactin-binding activity is also observed in subcellular fractions isolated from the adrenal gland, liver, ovary and kidney of the pregnant rabbit, a finding that is consistent with other reported actions of prolactin in these organs.     

Digestion by the larva of the pruinose scarab, Sericesthis geminata

An in vitro study of digestion by the subterranean larva of S. geminata revealed the presence of enzymes able to digest starch, trehalose, salicin, cellobiose, melibiose, maltose, sucrose, casein, and several forms of cellulose. The midgut is the major source of all enzymes except invertase and CMC-cellulase, for which hindgut preparations are more active. The pH values of the gut contents are: foregut 6.9, midgut 9.0 and hindgut 7.5. The midgut fluid is pumped forward into the foregut, and the carbohydrases in it are generally more active at the pH of the foregut than at the pH of the midgut. It is possible, therefore, that the foregut, which is itself insignificant as a source of enzymes, is the site of considerable carbohydrate digestion. The proteinase is inhibited 28% by trypsin inhibitors.

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Zusammenfassung Eine in vitro-Untersuchung über die Verdauungsvorgänge in den unterirdisch lebenden Larven von S. geminata wies Enzyme nach, die Stärke, Trehalose, Salicin, Cellobiose, Melibiose, Maltose, Rohrzucker, Kasein und mehrere Celluloseformen abbauen können. Versuche mit Vorder-, Mittel- und Enddarmextrakten zeigten, daß der Abbau von Rohrzucker und CMC-Cellulose in Ansätzen mit Enddarmpräparaten prozentual am höchsten war. Maximale Verdauung aller anderen geprüften Substrate erfolgte dagegen mit Mitteldarmpräparaten.Die pH-Werte betrugen im Vorderdarm 6,9, im Mitteldarm 9,0 und im Enddarm 7,5. Im Mittel- und Enddarm lagen die jewciligen optimalen pH-Werte für Maltose bei 6,5–7,0 und 7,5, für Melibiose bei 6,0–7,0 und 7,0–7,5, für Cellobiose bei 6,0–7,0 und 4,5, für Rohrzucker bei 7,0 und 6,0, für CMC bei 5,2 und 5,6.Die Verdauung unlöslicher Celluloseformen war durchweg gering, die der CMC dagegen beträchtlich.Die Mitteldarmflüssigkeit wird nach vorn in den Vorderdarm gepumpt. Die darin enthaltenen Carbohydrasen sind beim pH-Wert des Vorderdarmes meistens wirksamer als in dem des Mitteldarmes. Es ist deshalb möglich, daß im Vorderdarm, welcher selbst wahrscheinlich keine Enzyme produziert, doch eine beträchtliche Kohlenhydratverdauung stattfindet.Die Wirksamkeit der Proteinase wird durch bekannte Tryptin-Hemmstoffe um 28% reduziert.

     

Plant Regeneration from Isolated Microspore of Indica Rice

Isolated microspores of rice (Oryza saliva L.) cultivars, IR36and IR43, belonging to the recalcitrant indica subspecies werecultured. Two types of microspores were observed after isolationfrom the fresh anthers and from pre-cultured anthers—onetype consisted of vacuolated, larger-sized grains, while theother was composed of microspores of smaller sizes with densecytoplasm. Within few days in culture, all the smaller sizedgrains were dead, and only the large grains were viable andproduced pollen embryos. After 30 days from culture, microcalliwere transferred to semisolid modified Murashige and Skoog mediumcontaining 1 mg/liter each of kinetin and naphthaleneaceticacid and kept under continuous light at 25?C. IR36 showed onlycell division while IR43 gave 32 green plants from these experiments. (Received January 18, 1990; Accepted July 4, 1990)     

Characterization of a cDNA encoding cysteine proteinase inhibitor from Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower buds

A cDNA encoding a new phytocystatin isotype named BCPI-1 was isolated from a cDNA library of Chinese cabbage flower buds. The BCPI-1 clone encodes 199 amino acids resulting in a protein much larger than other known phytocystatins. BCPI-1 has an unusually long C-terminus. A BCPI-1 fusion protein expressed in Escherichia coli strongly inhibits the enzymatic activity of papain, a cysteine proteinase. Genomic Southern blot analysis revealed that the BCPI gene is a member of a small multi-gene family in Chinese cabbage. Northern blot analysis showed that it is differentially expressed in the flower bud, leaf and root.