Involvement of LFA-1 in lymphoma invasion and metastasis demonstrated with LFA-1-deficient mutants

Lymphocyte function-associated antigen-1 (LFA-1) is a leukocyte and lymphoma cell surface protein that promotes intercellular adhesion. We have previously shown that the invasion of hepatocyte cultures by lymphoma cells is inhibited by anti-LFA-1 antibodies (Roos, E., and F. F. Roossien. 1987. J. Cell Biol. 105:553-559). In addition, we now report that LFA-1 is also involved in invasion of lymphoma cells into fibroblast monolayers. To investigate the role of LFA-1 in metastasis of these lymphoma cells, we have generated mutants that are deficient in LFA-1 cell surface expression because of impaired synthesis of either the alpha or beta subunit precursor of LFA-1. We identified at least three distinct mutant clones. The invasive potential of the mutant cells in vitro, in both hepatocyte and fibroblast cultures, was considerably lower than that of parental cells. The metastatic potential of the mutants was much reduced, indicating that LFA-1 expression is required for efficient metastasis formation by certain lymphoma cells.     

Database on the structure of large ribosomal subunit RNA.

Our database on large ribosomal subunit RNA contained 334 sequences in July, 1995. All sequences in the database are aligned, taking into account secondary structure. The aligned sequences are provided, together with incorporated secondary structure information, in several computer-readable formats. These data can easily be obtained through the World Wide Web. The files in the database are also available via anonymous ftp.     

Database on the structure of small ribosomal subunit RNA.

The database on small ribosomal subunit RNA structure contains (June 1994) 2824 nucleotide sequences. All these sequences are stored in the form of an alignment based on the adopted secondary structure model, which in turn is corroborated by the observation of compensating substitutions in the alignment. The complete database is made available to the scientific community through anonymous ftp on our server in Antwerp. A special effort was made to improve electronic retrieval and a program is supplied that allows to create different file formats. The database can also be obtained from the EMBL nucleotide sequence library.     

13C nuclear magnetic resonance studies of Pseudomonas putida fatty acid metabolic routes involved in poly(3-hydroxyalkanoate) synthesis.

The formation of poly(3-hydroxyalkanoates) (PHAs) in Pseudomonas putida KT2442 from various carbon sources was studied by 13C nuclear magnetic resonance spectroscopy, gas chromatography, and gas chromatography-mass spectroscopy. By using [1-13C]decanoate, the relation between beta-oxidation and PHA formation was confirmed. The labeling pattern in PHAs synthesized from [1-13C]acetate corresponded to the formation of PHAs via de novo fatty acid biosynthesis. Studies with specific inhibitors of the fatty acid metabolic pathways demonstrated that beta-oxidation and de novo fatty acid biosynthesis function independently in PHA formation. Analysis of PHAs derived from [1-13C]hexanoate showed that both fatty acid metabolic routes can function simultaneously in the synthesis of PHA. Furthermore, evidence is presented that during growth on medium-chain-length fatty acids, PHA precursors can be generated by elongation of these fatty acids with an acetyl coenzyme A molecule, presumably by a reverse action of 3-ketothiolase.     

Both interleukin-1 alpha and interleukin-1 beta are involved as accessory signals in primary antigen (tetanus toxoid) induced human T-cell activation

The function of interleukin-1 alpha and interleukin-1 beta (IL-1 alpha, IL-1 beta) in tetanus toxoid (TT) induced T-cell proliferation in cultures of peripheral blood mononuclear cells (PBL) obtained from healthy donors was assessed by using neutralizing antisera to IL-1 alpha and IL-1 beta. The neutralizing capacity and the specificity of the IL-1 antisera were tested by the use of the thymoma EL-4 NOB-1 cell line. Antisera to IL-1 beta effectively neutralized the proliferative capacity of human recombinant IL-1 beta but not of human recombinant IL-1 alpha and vice versa. Addition of either anti-IL-1 beta or anti-IL-1 alpha antiserum to the culture medium hardly affected TT induced T-cell proliferation. However, the proliferative T-cell response was consistently attenuated when a combination of IL-1 alpha and IL-1 beta antiserum was used. The antisera were never capable of completely abolishing the T-cell response to TT. We conclude that (a) IL-1 alpha and IL-1 beta are both necessary accessory signals for T-cell proliferation to antigen in vitro; (b) in T-cell proliferation IL-1 alpha and IL-1 beta are interchangeable; and (c) T-cell proliferation to antigen is only partially dependent on IL-1 as signal.     

Analysis of docosahexaenoic acid biosynthesis in Crypthecodinium cohnii by 13C labelling and desaturase inhibitor experiments

The lipids of the heterotrophic microalga Crypthecodinium cohnii contain the omega-3 polyunsaturated fatty acid (PUFA) and docosahexaenoic acid (22:6) to a level of over 30%. The pathway of 22:6 synthesis in C. cohnii is unknown. The ability of C. cohnii to use 13C-labelled externally supplied precursor molecules for 22:6 biosynthesis was tested by 13C NMR analysis. Furthermore, the presence of desaturases (typical for aerobic PUFA synthesis) was studied by the addition of specific desaturase inhibitors in the growth medium. The addition of 1-(13)C acetate or 1-(13)C butyrate in the growth medium resulted in 22:6 with only the odd carbon atoms enriched. Apparently, two-carbon units were used as building blocks for 22:6 synthesis and butyrate was first split into two-carbon units prior to incorporation in 22:6. When 1-(13)C oleic acid was added to the growth medium, 1-(13)C oleic acid was incorporated into the lipids of C. cohnii but was not used as a precursor for the synthesis of 22:6. Specific desaturase inhibitors (norflurazon and propyl gallate) inhibited lipid accumulation in C. cohnii. The fatty acid profile, however, was not altered. In contrast, in the arachidonic acid-producing fungus, Mortierella alpina, these inhibitors not only decreased the lipid content but also altered the fatty acid profile. Our results can be explained by the presence of three tightly regulated separate systems for the fatty acid production by C. cohnii, namely for (1). the biosynthesis of saturated fatty acids, (2). the conversion of saturated fatty acids to monounsaturated fatty acids and (3). the de novo synthesis of 22:6 with desaturases involved.     

Nuclear staining for the small heat shock protein alphaB-crystallin colocalizes with splicing factor SC35

AlphaB-Crystallin has for a long time been considered a specific eye lens protein. Later on it appeared that this protein belongs to the family of the small heat shock proteins and that it occurs also extra-lenticularly in many different cell types. AlphaB-Crystallin is mainly present in the cytoplasm, but there are some indications that it might have a function in the nucleus too. However, till now its presence in the nucleus is uncertain. We therefore compared the localization of alphaB-crystallin in nine cell lines cultured under normal conditions using four different antisera. All four antisera gave a diffuse staining for alphaB-crystallin in the cytoplasm, but one of the antibodies consistently showed nuclear staining in eight of the cell types, in the form of distinct speckles. These speckles are equally pronounced in the different cell types, whether or not cytoplasmic alphaB-crystallin is present. Preabsorption of the antiserum with alphaB-crystallin abolished the staining. Furthermore we demonstrate that if only minor amounts of alphaB-crystallin are present, the protein seems to be located exclusively in the nucleus. However, in case of higher amounts of protein, alphaB-crystallin is distributed between cytoplasm and nucleus. The nuclear alphaB-crystallin exists, like the cytoplasmic alphaB-crystallin, in non-phosphorylated and phosphorylated forms, is Triton-insoluble but can be extracted by 2 M NaCl. These data suggest that alphaB-crystallin might be bound to the nuclear matrix per se or to nuclear matrix proteins via other proteins. In agreement with other nuclear matrix proteins, nuclear alphaB-crystallin staining turns diffuse upon mitosis and leaves the chromosomes unstained. Double staining experiments revealed colocalization of alphaB-crystallin with the splicing factor SC35 in nuclear speckles, suggesting a role for alphaB-crystallin in splicing or protection of the splicing machinery.     

Cigarette smoke-induced blockade of the mitochondrial respiratory chain switches lung epithelial cell apoptosis into necrosis

Increased lung cell apoptosis and necrosis occur in patients with chronic obstructive pulmonary disease (COPD). Mitochondria are crucially involved in the regulation of these cell death processes. Cigarette smoke is the main risk factor for development of COPD. We hypothesized that cigarette smoke disturbs mitochondrial function, thereby decreasing the capacity of mitochondria for ATP synthesis, leading to cellular necrosis. This hypothesis was tested in both human bronchial epithelial cells and isolated mitochondria. Cigarette smoke extract exposure resulted in a dose-dependent inhibition of complex I and II activities. This inhibition was accompanied by decreases in mitochondrial membrane potential, mitochondrial oxygen consumption, and production of ATP. Cigarette smoke extract abolished the staurosporin-induced caspase-3 and -7 activities and induced a switch from epithelial cell apoptosis into necrosis. Cigarette smoke induced mitochondrial dysfunction, with compounds of cigarette smoke acting as blocking agents of the mitochondrial respiratory chain; loss of ATP generation leading to cellular necrosis instead of apoptosis is a new pathophysiological concept of COPD development.