Characterization of laminin isoforms in human amnion

Epithelial cells of the human amnion have been reported to possess similar functions to many types of cells, such as hepatocytes, neurons, and pancreatic beta-cells. We reported previously that one of the hepatocyte-like functions of human amniotic epithelial cells was reinforced by the presence of basement membrane components. Laminin is one of the main components of the basement membrane; it critically contributes to cell differentiation. Laminin has several heterotrimer isoforms composed of an alpha-, a beta-, and a gamma-chain, and each type of chain has several types of subunit chains: alpha1-5, beta1-3, and gamma1-3. In this study, we characterized the laminin subunit chains in human amnion. Laminin is produced and secreted from adjacent epithelial cells, and therefore, the gene expression of laminin subunit chains in human amniotic epithelial cells was investigated by RT-PCR. Their localization was examined by immunohistochemical staining of frozen sections. The findings suggested that the basement membrane of the human amnion contains a broad spectrum of laminin isoforms, laminin-2, -4, -5, -6, -7, -10, -11. These findings will provide clues not only for understanding the physiological roles of the amnion and hAECs, but also for applying this tissue as a source of donor cells for cell transplantation therapy.     

Cytosolic chaperonin-containing t-complex polypeptide 1 changes the content of a particular subunit species concomitant with substrate binding and folding activities during the cell cycle.

The chaperonin-containing t-complex polypeptide 1 (CCT) is a cytosolic molecular chaperone composed of eight subunits that assists in the folding of actin, tubulin and other cytosolic proteins. We show here that the content of particular subunits of CCT within mammalian cells decreases concomitantly with the reduction of chaperone activity during cell cycle arrest at M phase. CCT recovers chaperone activity upon resumption of these subunits after release from M phase arrest or during arrest at S phase. The levels of alpha, delta and zeta-1 subunits decreased more rapidly than the other subunits during M phase arrest by colcemid treatment and recovered after release from the arrest. Gel filtration chromatography or native (nondenaturing) PAGE analysis followed by immunoblotting indicated that the alpha and delta subunit content in the 700- to 900-kDa CCT complex was appreciably lower in the M phase cells than in asynchronous cells. In vivo, the CCT complex of M-phase-arrested cells was found to bind lower amounts of tubulin than that of asynchronous cells. In vitro, the CCT complex of M phase-arrested cells was less active in binding and folding denatured actin than that of asynchronous cells. On the other hand, the CCT complex of asynchronous cells (a mixture of various phases of cell cycle) exhibited lower alpha and delta subunit content and lower chaperone activity than that of S-phase-arrested cells obtained by excess thymidine treatment. In addition, turnover (synthesis and degradation) rates of the alpha and delta subunits in vivo were more rapid than those of most other subunits. These results suggest that the content of alpha and delta subunits of CCT reduces from the complete active complex in S phase cells to incomplete inactive complex in M phase cells.     

Identification and characterization of lactic acid bacteria in ragi tape

One hundred and eighteen lactic acid bacteria (LAB) were isolated from five different types of ragi tape, a traditional dry-starter of Balinese rice wine. The isolates could be classified into three groups based on the cell shape and capability to produce gas from glucose. Group I contained 66 homofermentative cocci, group II contained seven homofermentative rods, and group III contained 45 heterofermentative rods. Among these 118 isolates, 21 isolates representing these groups were selected and were first identified using phenotypic characters. The identification performed phenotypically was confirmed by sequencing of variable region 8 (V8) of the 16S rDNA. The comparative studies led to the identification of Pediococcus pentosaceus, Enterococcus faecium, Lactobacillus curvatus, Weissella confusa, and W. paramesenteroides from the ragi tape examined.     

Role of FK506-binding protein 12 in development of the chick embryonic heart

A cDNA encoding chicken FK506-binding protein 12 (FKBP12) was isolated and sequenced. The predicted amino acid sequence of the chicken protein shows high homology to those of FKBP12 proteins of other species ranging from human to frog. The possible role of FKBP12 in chick embryonic cardiac development was examined. Northern blot analysis revealed that FKBP12 mRNA is distributed widely in chick embryos, being especially abundant in the heart; the amount of FKBP12 mRNA in the embryonic heart decreased with time. Administration of FK506 to chick embryos at 7 to 9 days resulted in marked cardiac enlargement. FK506 also reduced the expression of myosin, induced a more elongated cell morphology, and impaired network formation in cultured chick embryonic cardiomyocytes. These results suggest that FKBP12 is important in the regulation of contractile function and phenotypic expression in chick cardiomyocytes during embryonic development.     

Cutting edge: IL-18-transgenic mice: in vivo evidence of a broad role for IL-18 in modulating immune function

IL-18 has been shown to be a strong cofactor for Th1 T cell development. However, we previously demonstrated that when IL-18 was combined with IL-2, there was a synergistic induction of a Th2 cytokine, IL-13, in both T and NK cells. More recently, we and other groups have reported that IL-18 can potentially induce IgE, IgG1, and Th2 cytokine production in murine experimental models. Here, we report on the generation of IL-18-transgenic (Tg) mice in which mature mouse IL-18 cDNA was expressed. CD8+CD44high T cells and macrophages were increased, but B cells were decreased in these mice while serum IgE, IgG1, IL-4, and IFN-gamma levels were significantly increased. Splenic T cells in IL-18 Tg mice produced higher levels of IFN-gamma, IL-4, IL-5, and IL-13 than control wild-type mice. Thus, aberrant expression of IL-18 in vivo results in the increased production of both Th1 and Th2 cytokines.     

Identification of a human homologue of the dendritic cell-associated C-type lectin-1, dectin-1

Previously we identified the novel type II lectin receptor, dectin-1, that is expressed preferentially by murine antigen presenting dendritic cells (DC) and is involved in co-stimulation of T cells by DC. To identify the human homologue (DECTIN-1), we employed degenerative PCR amplification of mRNA isolated from DC and subsequent cDNA cloning. DECTIN-1 is a type II lectin receptor with high homology to type II lectin receptors expressed by natural killer (NK) cells. It contains an immunoreceptor tyrosine-based activation motif within the cytoplasmic domain. Human DECTIN-1 mRNA is expressed predominantly by peripheral blood leukocytes and preferentially by DC. The mRNA likely encodes a 33 kDa glycoprotein. In human epidermis, the protein is expressed selectively by Langerhans cells, which are an epidermal subset of DC. A truncated form of DECTIN-1 RNA (termed T beta) encodes for a polypeptide lacking almost the entire neck domain, which is required for accessibility of the carbohydrate recognition domain to ligands. Genome analysis showed the deleted amino acid sequence in T beta to be encoded by an exon, indicating that T beta RNA is produced by alternative splicing. DECTIN-1 gene maps to chromosome 12, between p13.2 and p12.3, close to the NK gene complex (12p13.1 to p13.2) which contains genes for NK lectin receptors. Our results indicate that human DECTIN-1 shares many features with mouse dectin-1, including the generation of neck domain-lacking isoforms, which may down-regulate the co-stimulatory function of dectin-1.     

Mechanism of selective inhibition of respiratory syncytial virus by a benzodithiin compound (RD3-0028)

RD3-0028, a compound with a benzodithiin structure, was found to be a potent inhibitor of respiratory syncytial virus (RSV) replication. Its action is specific; no activity is seen against influenza A virus, measles virus, herpes simplex virus type 1 or 2, or human cytomegalovirus. A time-dependent drug addition experiment indicated that the antiviral activity occurs in the late stage of the RSV replication cycle, since this compound completely inhibited syncytium formation even when added up to 16 hr after the infection of cell monolayers at an MOI of 3. RD3-0028 had no direct virucidal effect on RSV. Western blotting analysis showed that RD3-0028 significantly decreased the amount of RSV proteins released into the cell culture medium. Moreover, five independent isolates of the RSV long strain were selected for growth in RD3-0028 (5-20 microg/ml). These resistant viruses were more than 80-fold less sensitive to RD3-0028 than the long strain. The F gene segment of each of these viruses was sequenced and in each case the mutant RNA segment contained at least one sequence alteration, converting asparagine 276 to tyrosine (F1 protein). These results suggest that RD3-0028 inhibits RSV replication by interfering with intracellular processing of the RSV fusion protein, or a step immediately thereafter, leading to loss of infectivity.     

Reactivities of mutants of a major house dust mite allergen Der f 2 to mouse anti-Der f 2 monoclonal antibodies analyzed by immunoblotting

A total of sixteen recombinant variants of a major house dust mite allergen Der f 2, the wild-type Der f 2, six cysteine mutants, six proline mutants, and three lysine mutants, were expressed in Escherichia coli. The cells were solubilized and run on SDS-PAGE under reducing conditions. Epitopes for five mouse anti-Der f 2 monoclonal antibodies, 1B2, 7C10, 13A4, 15E11, and 18G8, to the recombinant Der f 2 variants were characterized by immunoblot analysis.     

Production of clover broomrape seed germination stimulants by red clover root requires nitrate but is inhibited by phosphate and ammonium

The effect of nutrients (nitrate, ammonium, urea, phosphate and potassium) on the production and/or exudation of germination stimulants for clover broomrape ( Orobanche minor Sm.), a root holoparasite, by its host red clover ( Trifolium pratense L.) was examined using hydroponically grown material. Potassium (K₂SO₄) concentrations up to 100 mg l^–1 (based on K) did not affect the production of germination stimulants by red clover while, in contrast, phosphate (NaH₂PO₄) was highly inhibitory even at concentrations as low as 1 mg l^–1 (based on P). Nitrate (NaNO₃) markedly promoted stimulant production in a dose-dependent manner from 2 to 50 mg l^–1 (based on N). Ammonium [(NH₄)₂SO₄] had no effect at 2 mg l^–1 (based on N) but was inhibitory at higher concentrations. Ammonium is known to be a seed germination inhibitor of root parasites, indicating that ammonium has a dual inhibitory action. Urea had no effect at 2 mg l^–1 (based on N) but was promotive at higher concentrations. These results provide a basis for the inhibitory effects of nitrogen fertilizer on infection by root parasitic weeds, broomrapes and witchweeds, and explain why these parasites prevail in areas where there is lower phosphorus availability in soils.     

Accumulation of 6-deoxocathasterone and 6-deoxocastasterone in Arabidopsis, pea and tomato is suggestive of common rate-limiting steps in brassinosteroid biosynthesis

To gain a better understanding of brassinosteroid biosynthesis, the levels of brassinosteroids and sterols related to brassinolide biosynthesis in Arabidopsis, pea, and tomato plants were quantified by gas chromatography-selected ion monitoring. In these plants, the late C-6 oxidation pathway was found to be the predominant pathway in the synthesis of castasterone. Furthermore, all these plant species had similar BR profiles, suggesting the presence of common biosynthetic control mechanisms. The especially high levels of 6-deoxocathasterone and 6-deoxocastasterone may indicate that their respective conversions to 6-deoxoteasterone and castasterone are regulated in planta and hence are important rate-limiting steps in brassinosteroid biosynthesis. Other possible rate-limiting reactions, including the conversion of campestanol to 6-deoxocathasteonre. are also discussed. Tomato differs from Arabidopsis and pea in that tomato contains 28-norcastasterone as a biologically active brassinosteroid, and that its putative precursors, cholesterol and its relatives are the major sterols.