Expression of a novel human ornithine decarboxylase-like protein in the central nervous system and testes.

Ornithine decarboxylase (ODC) is the key enzyme of polyamine synthesis. The physiological activity of ODC is associated with cell proliferation, and high ODC activities are encountered in rapidly growing cancer cells. We have cloned a cDNA for a novel human protein that is 54% identical to ODC and 45% identical to antizyme inhibitor (AZI). mRNA for ODC-paralogue (ODC-p) was found only in the central nervous system and testes, suggesting a role in terminal differentiation rather than cell proliferation. ODC-p occurs at least in eight alternatively spliced forms. In vitro translated ODC-p did not decarboxylate ornithine, whereas, in vivo, one splice variant exerted modest ODC-like activity upon expression in COS-7 cells. ODC-p has a unique mutation in cysteine 360, where this ornithine decarboxylase reaction-directing residue is substituted by a valine. This substitution might lead to an enzymatic reaction that differs from typical ODC activity. ODC-p might also function as a brain- and testis-specific AZI.

The roles of claudin superfamily proteins in paracellular transport.

The claudin superfamily consists of at least 18 homologous proteins in humans. These proteins are important structural and functional components of tight junctions in paracellular transport. Complexed with two other integral transmembrane proteins, occludin and junctional adhesion molecule, claudins are located in both epithelial and endothelial cells in all tight junction-bearing tissues. Claudins interact directly with tight junction-specific, membrane-associated guanylate kinase homologues, ZO-1, ZO-2, and ZO-3, and indirectly with AF-6 and the myosin-binding molecule cingulin. These protein-protein interactions promote scaffolding of the tight junction transmembrane proteins and provide a link to the actin cytoskeleton for transducing regulatory signals to and from tight junctions. The distinct permeability properties observed in different epithelia and endothelia seemingly result from the restricted tissue expression, variability of the homopolymer and heteropolymer assembly, regulated transcription and translation, and the subcellular localization of claudin family proteins. Defects in claudins are causatively associated with a variety of human diseases, demonstrating that claudins play important roles in human physiology. In conditions where the cell adhesion function contributed by tight junctions is essential, such as in altered paracellular transport, in proliferative diseases, and during morphogenesis, the claudin superfamily of homologous proteins provides the molecular basis for the uniqueness of tight junctions and emerges as a new target for intervention.

The roles of iron in health and disease.

Iron is vital for almost all living organisms by participating in a wide variety of metabolic processes, including oxygen transport, DNA synthesis, and electron transport. However, iron concentrations in body tissues must be tightly regulated because excessive iron leads to tissue damage, as a result of formation of free radicals. Disorders of iron metabolism are among the most common diseases of humans and encompass a broad spectrum of diseases with diverse clinical manifestations, ranging from anemia to iron overload and, possibly, to neurodegenerative diseases. The molecular understanding of iron regulation in the body is critical in identifying the underlying causes for each disease and in providing proper diagnosis and treatments. Recent advances in genetics, molecular biology and biochemistry of iron metabolism have assisted in elucidating the molecular mechanisms of iron homeostasis. The coordinate control of iron uptake and storage is tightly regulated by the feedback system of iron responsive element-containing gene products and iron regulatory proteins that modulate the expression levels of the genes involved in iron metabolism. Recent identification and characterization of the hemochromatosis protein HFE, the iron importer Nramp2, the iron exporter ferroportin1, and the second transferrin-binding and -transport protein transferrin receptor 2, have demonstrated their important roles in maintaining body's iron homeostasis. Functional studies of these gene products have expanded our knowledge at the molecular level about the pathways of iron metabolism and have provided valuable insight into the defects of iron metabolism disorders. In addition, a variety of animal models have implemented the identification of many genetic defects that lead to abnormal iron homeostasis and have provided crucial clinical information about the pathophysiology of iron disorders. In this review, we discuss the latest progress in studies of iron metabolism and our current understanding of the molecular mechanisms of iron absorption, transport, utilization, and storage. Finally, we will discuss the clinical presentations of iron metabolism disorders, including secondary iron disorders that are either associated with or the result of abnormal iron accumulation.

Translocation of ornithine decarboxylase to the surface membrane during cell activation and transformation.

Ornithine decarboxylase (ODC) is highly up-regulated in proliferating and transforming cells. Here we show that upon induction, an initial cytosolic increase of ODC is followed by translocation of a fraction of the enzyme to the surface membrane. ODC membrane translocation is mediated by a p47(phox) membrane-targeting motif-related sequence, as indicated by reduced ODC activity in the membrane fraction of cells treated with a competing, ODC-derived (amino acids 165-172) peptide, RLSVKFGA, which is homologous to the p47(phox) membrane-targeting sequence. p47(phox) membrane translocation is known to be dependent on the phosphorylation of the targeting motif. Analogously, overexpressed ODC.S167A, a mutant ODC lacking the putative phosphorylation site Ser67, is unable to move to the surface membrane. Cells blocked with the RLSVKFGA peptide showed defective transformation, indicating that the motif-mediated translocation of ODC is prerequisite to its biological function. Constitutive targeting of ODC to the membrane using a plasmid encoding the chimeric protein, wild-type ODC with C-terminal linkage to the farnesylation motif of K-ras, caused impaired cytokinesis with an accumulation of polykaryotic cells. Impaired cytokinesis confirms that ODC is involved in mitotic cytoskeletal rearrangement events and pinpoints the importance of relevant membrane targeting to its physiological function.

Activation of natural killer cells via the Fc gamma RIII (CD16) requires initial tyrosine phosphorylation.

Triggering of the Fc gamma RIII (CD16) on natural killer (NK) cells by monoclonal antibodies or antibody-coated target cells stimulates a rapid phospholipase C (PLC)-mediated hydrolysis of inositol phospholipids and results in subsequent delivery of the lytic hit. The role of initial tyrosine phosphorylation in these events was investigated with a tyrosine protein kinase (TPK) inhibitor, genistein. At doses that inhibited CD16-triggered tyrosine phosphorylation of substrates in intact cells, genistein did not influence serine/threonine phosphorylation or target cell binding but prevented PLC activation, cell-mediated cytotoxicity and antibody-dependent cellular cytotoxicity. These findings indicate that tyrosine phosphorylation is an early and critical event during receptor-mediated activation of the lytic machinery.

Role of CD16 (Fc receptor III) and interleukin-2 in the reactivation of natural killer cells after inhibitory target cell contact.

Contact with natural killer (NK)-resistant monolayer targets is an inhibitory signal to NK cells. In this study, we have analyzed the effect of such effector/target cell interactions on the CD16 (FcRIII) expression on lymphocytes and the role of CD16 and interleukin-2 (IL-2) in the reactivation of their cytolytic machinery. Coculturing peripheral blood mononuclear cells with NK-resistant monolayer cells did not change the percentage of CD 16-positive effector cells, although this treatment effectively inhibited their cytotoxicity against NK-sensitive targets. The inhibited effector cells partially regained their activity by incubating for 24 h in medium supplemented with 10% fetal calf serum (FCS), whereas human albumin-, newborn calf serum- or human AB serum-supplemented media had no reactivating effect. Monoclonal class IgG1, IgG2a and IgM anti-CD16 antibodies [Abs; 3G8, CLB-CD16 (CLB-FcR gr1) and Leu 11b], and normal rabbit IgG (NR-IgG) prevented the FCS-mediated reactivation of cytotoxicity, whereas nonreactive control Abs significantly enhanced it. The detection of the CD16 antigen by the monoclonal anti-CD16 Abs Leu 11a and Leu 11c was blocked by the above anti-CD16 Abs and NR-IgG, while the expression of other NK cell-associated surface molecules (CD2, CD56) remained unchanged. Mere blocking of CD16, using a short-term incubation with anti-CD16 Abs, had an insignificant effect on endogenous NK activity, suggesting that CD16 is involved in NK cell (re)activation rather than in the killing process itself. In the presence of IL-2, inactivated effector cells also regained their killing activity. The IL-2-induced reactivation was not inhibited by anti-CD16 Abs. The results suggest that FCS-derived factors and soluble nonreactive immunoglobulins enhance the NK activity of down-regulated effector cells via CD16, and that CD16 and IL-2 receptors represent alternative independent pathways of NK cell reactivation.

Participation of CD11a-c/CD18 and RGD-recognizing adhesion molecules in the binding of LGL to fibroblasts. Evidence for the role of CD11a in the fibroblast-mediated inactivation of NK cells.

We have previously shown that large granular lymphocytes (LGL) are inactivated by contact with natural killer (NK) resistant monolayer target cells. In this work we have analysed which adhesion molecules are involved in the binding of LGL to such targets, as exemplified by fibroblasts, and in the subsequent inhibition of their NK activity. The results indicate that antibodies against CD54 (intercellular adhesion molecule 1, ICAM-1), CD11a (leucocyte function antigen 1, LFA-1, alpha chain), and CD18 (common beta chain of the beta 2-integrin family) significantly (by 50%) reduce the binding of LGL onto inhibitory target cells. The matrix protein-based synthetic peptide RGD and anti-CD29 (the common beta chain of the beta 2-integrin family) antibodies also diminish the binding (by 35%). The effects of the antiadhesion molecule antibodies and the peptide are additive, the combination of both leading to an almost complete block of adhesion. It may be hypothesized that some of the binding-relevant adhesion molecules of the RGD-binding domain on LGL (CD29) may be involved in the delivery of the inactivating signal to the effector cell. Indeed, incubation of LGL with anti-CD11a antibodies, but neither with antibodies against other binding-relevant epitopes nor with RGD, significantly reduced their NK activity. The mechanism of the inactivation was similar to that induced by intact NK-resistant target cells. On the basis of the present results we suggest that the CD11a molecule is involved in the down-regulation of the NK activity of peripheral blood lymphocytes.

Characteristics of soluble tumour-derived proteins that inhibit natural killer activity.

We have previously shown that various benign and malignant natural killer (NK)-resistant monolayer cells inhibit endogenous human NK activity, probably by reducing the secretion of cytotoxic factors from the effector cells. The nature of the molecules responsible for the inhibition has been unclear. In this study we show that phosphate-buffered saline (PBS) extracts of ovarian cystadenocarcinoma tissue and normal uterine smooth muscle strongly inhibit NK activity. Fractionation of tumour extracts by gel chromatography revealed major inhibitory activity in the Mr range 160,000-180,000, and other weaker inhibiting activities in the Mr ranges 50,000-70,000 and 20,000. The active material of Mr range 160,000-180,000 was adsorbed on anion exchange chromatography column at neutral pH and physiologic NaCl concentration, and it was eluted by 0.31-0.34 M NaCl. The inhibitory molecule was sensitive to proteolysis. No relation of this compound to immunoglobulins or trypsin and urokinase inhibitors was detected. The unfractionated extract inhibited NK activity apparently by the same mechanism as the monolayer target cells, i.e. by reducing the secretory capacity of effector cells. The data strongly suggest that the NK-inhibiting compounds described in this work are involved in the inactivation of NK cells by intact monolayer cells.

Effect of interleukin 2 on the inhibition of human natural killer activity by monolayer cells.

We have previously shown that human endogenous natural killer activity against K562 is inhibited by primary cultures of natural killer-resistant monolayer target cells. In this study we have analyzed the sensitivity of activated killer cells to this inhibitory effect. Interleukin-2 (IL-2), when present during an 18-hr contact of peripheral blood lymphocytes with monolayers, did not affect the inhibition of natural killer cell activity. Pretreatment of effector cells with IL-2 for 24-62 hr before the contact with monolayer cells eradicated the inhibition caused by malignant cells, benign cells remaining inhibitory. The IL-2-pretreated effector cells killed preferentially malignant target cells, although significant cytotoxicity was also detectable against benign cell cultures. The results indicate that activation of killer cells in vitro by IL-2 involves the desensitization of effector cells to the inhibitory signals of target cells, and that the selectivity of IL-2-activated killer cells toward malignant target cells involves weaker inhibition of activated killer cells by malignant cells.

Mechanism of cell contact-mediated inhibition of natural killer activity.

Natural killer cell activity is inhibited by primary cultures of monolayer cells. In this study, we analyzed the mechanism of the inhibition. Inhibited NK cells showed unaltered binding capacity to NK sensitive K562 cells. The orientation of the effector cells' actin-containing microfilaments, an event known to occur during the programming for the lysis stage in lytic conjugates, was unaffected by the inhibition. In single cell cytotoxicity experiments, the number of killer cells among conjugate-forming cells was reduced. The capacity of the inactivated NK cells to secrete cytotoxic factors upon stimulation with Con A was also impaired. Both NK-resistant inactivating target cells and NK-sensitive K562 cells were sensitive to the toxic factors secreted by NK cells. Thus, the results indicate that the target cell-mediated inactivation of NK cell is based on a block in the lethal hit stage, possibly due to reduced release of toxic factor(s) from the effector cells. The capacity of inactivated effector cells to mediate antibody-dependent cellular cytotoxicity was unimpaired, suggesting that the contact-mediated inhibition of cytotoxicity selectively affects NK cells.

Non-Hodgkin's lymphomas in childhood. A clinicopathologic and epidemiologic study in Finland.

All cases diagnosed in Finland as non-Hodgkin's lymphoma (NHL), Hodgkin's disease or histiocytosis X in children younger than 15 years in 1953 to 1973, according to the Finnish Cancer Registry, were reexamined histologically. Only 55% of the cases originally diagnosed as NHL were regarded as such at reexamination. The others were mainly malignant nonlymphatic tumors such as neuroblastoma and different kinds of sarcomas. Seventy-two NHLs were diagnosed in 50 boys and 22 girls. The corrected age-specific incidence rate was 0.32/10(5). The most common histologic types were Burkitt's lymphoma (BL) (30 cases), lymphoblastic lymphoma (LBL) (26), large cell lymphomas (LCL) (six), and non-Burkitt's lymphoma (n-BL) (three). There were marked differences between BL and LBL in the course of the disease: BL was extranodal in 83%, LBL only in 4% (mediastinum was regarded as nodal); BL showed initial abdominal or pelvic involvement in 60% whereas LBL showed none; BL had initial mediastinal involvement in 7%, and LBL had it in 62%; all patients with LBL died whereas 23% of those with BL survived. Other types of NHL resembled BL in their course of disease. Patients with initial tonsillary involvement appeared to have the best prognosis and patients with mediastinal involvement the poorest. The importance of accurate histologic classification is emphasized. It appears to be most important to differentiate LBL from other types of NHL.

Effect of interferons on the inhibition of human natural killers by primary monolayer cell cultures.

Natural killer (NK) activity is inhibited by the contact of peripheral blood lymphocytes with primary monolayer cell cultures of both benign and malignant origin. In this study the effect of interferons (IFNs) on the inhibition has been analysed. Both alpha IFN- and gamma IFN-pretreated peripheral blood lymphocytes are effectively inhibited by monolayer target cells. IFN treatment of lymphocytes does not change cytotoxicity against the inhibitory target cells, although it enhances reactivity against NK-sensitive target cells. Treatment of monolayer cells with interferons significantly reduces their inhibitory capacity. However, diminished inhibition of NK activity by the IFN-treated target cells is not associated with increased lysis, probably due to their decreased sensitivity to natural killer cytotoxic factors (NKCF). In 18.5% of the cases studied, monolayer target cells induced endogenous IFN production in lymphocytes. In these cases no inhibition of the NK activity of the effector cells was seen. According to the results of this paper, IFNs have a dual effect on the NK regulatory system: they enhance the NK activity of the effector cells against NK-sensitive target cells, and they change the NK resistant target cells in a way that makes them less inhibitory to NK activity but simultaneously more resistant to the toxic factors secreted by NK cells.

Inhibition of human natural killer activity by monolayers of primary cell cultures.

Some primary and continuous cell cultures were tested for their capacity to regulate human natural killer (NK) activity. Primary cultures of endothelial cells, fetal fibroblasts, adult fibroblasts, amnion epithelial cells, renal parenchymal cells, and ovarian carcinoma cells inhibited NK activity when peripheral blood lymphocytes were preincubated on target cell monolayers for 18 h before testing the cytotoxicity against K-562. The supernatants of the inhibiting cell cultures were not suppressive. Prostaglandins or suppressive lymphocytes were not involved in the phenomenon. The binding capacity of the effector cells was not changed, suggesting that the suppressive signal was targeted at the cytolytic machinery of NK cells. The down-regulating capacity of the cell cultures weakened significantly during subculturing in vitro, and continuous cell lines were not inhibitory. The inactivation of NK cells may be one of the mechanisms by which target cells are protected from NK activity.

A new method for testing tuberculin skin reactivity--chamber test.

The Mantoux test and a chamber tuberculin test applied to the surface of the skin in 4 concentrations were performed on 229 children and 516 adults. The results were recorded at 72 hours. There was a significant correlation between the two tests. The chamber tuberculin test is technically easy, painless and atraumatic. It gives an opportunity of using a full range of concentrations of tuberculin resulting in a quantitative measurement of sensitivity in one and the same test procedure.

Skin prick test reactivity to common allergens in Finnish adolescents.

We studied 708 adolescents aged 15-17 years in the 9th grade of school in Imatra. Of the eligible population born in 1962 77% were included. All were skin prick tested with 16 extracts from two manufacturers with 12 common allergens, which included pollens, epithelia, mite, house dust and fish. At least one positive, immediate reaction (weal diameter 3 mm or larger) occurred in 49% and at least two positive reactions in 43% of those studied. The boys were observed to be significantly more reactive than the girls. The allergen preparations to which positive reactions were most prevalent were house dust, cat and horse epithelium, and mite extract. Large differences in the prevalence of positive reactions were observed with different preparations of the same allergen. Pollen allergens tended to cause the largest positive weal reactions, and the weal size distribution with some pollens was distinctly bimodal. A scheme for calculating allergen potency in histamine-equivalent-prick (HEP) units is presented. It is noted that the result is greatly dependent on the population group chosen for testing.

Allergic disorders and immediate skin test reactivity in Finnish adolescents.

We studied the prevalence of allergic disorders in an unselected group of 708 adolescents aged 15-17 years. All subjects were physically examined and interviewed by the authors. The prevalence of past or present asthma was 5.7% in boys and 3.1% in girls. The figures for hay fever were 14% and 8%, and for atopic dermatitis (including allergic urticaria) 25% and 30%, respectively. In 24% of all symptomatic subjects, the condition had not been active during the year preceding the study. The sex difference in the prevalence of hay fever was significant. It is associated with higher immediate skin test reactivity in boys. A progressive increase in the frequency of allergic disorders was observed with increasing number of positive skin reactions in both boys and girls. Respiratory allergy was closely related to a positive skin test: 87% of the asthmatics and 83% of all those with allergic rhinitis exhibited at least one positive skin reaction. For atopic dermatitis the association was less pronounced. Nineteen per cent of the population studied had a positive symptom history and a positive skin test to pollens, animal epithelia or dusts indicating a clinically significant relationship. However, 39% of the 346 subjects with a positive skin test, including some with a large number of positive reactions, were completely asymptomatic.

Plasma free tryptophan variation during oestrous cycle of the rat.

The concentration of plasma free tryptophan was shown to undergo a regular and statistically significant fluctuation through the four phases of the oestrous cycle of the rat. The highest concentration of plasma free tryptophan was observed during oestrus. During metoestrus the concentration of plasma free tryptophan decreased by 25% to the lowest observed level; thereafter it gradually increased during dioestrus and pro-oestrus. The concentration of plasma total tryptophan was found to be unchanged during the oestrous cycle of the rat.