Assigning functions to newly discovered genes constitutes one of the major challenges en route to fully exploiting the data becoming available from the genome sequencing initiatives. Heterologous expression in an appropriate host is central in functional genomics studies. In this context, filamentous fungi offer many advantages over bacterial and yeast systems. To facilitate the use of filamentous fungi in functional genomics, we present a versatile cloning system that allows a gene of interest to be expressed from a defined genomic location of Aspergillus nidulans. By a single USER cloning step, genes are easily inserted into a combined targeting-expression cassette ready for rapid integration and analysis. The system comprises a vector set that allows genes to be expressed either from the constitutive PgpdA promoter or from the inducible PalcA promoter. Moreover, by using the vector set, protein variants can easily be made and expressed from the same locus, which is mandatory for proper comparative analyses. Lastly, all individual elements of the vectors can easily be substituted for other similar elements, ensuring the flexibility of the system. We have demonstrated the potential of the system by transferring the 7,745-bp large mpaC gene from Penicillium brevicompactum to A. nidulans. In parallel, we produced defined mutant derivatives of mpaC, and the combined analysis of A. nidulans strains expressing mpaC or mutated mpaC genes unequivocally demonstrated that mpaC indeed encodes a polyketide synthase that produces the first intermediate in the production of the medically important immunosuppressant mycophenolic acid.

Expression of the receptor for the urokinase type plasminogen activator (uPAR) has been studied by flow cytometry and immunohistology in normal blood and bone marrow cells, in vitro activated lymphoid cells, and tissue samples from reactive lymph nodes (n = 6), thymus (n = 2) and malignant lymphomas (n = 82), or leukemias (n = 32). HL-60 myeloid precursor cells and CD34-positive normal stem cells also were analyzed. In the normal cells, staining was confined to monocytes, macrophages, neutrophils, and myeloid precursors. No labelling was seen of normal or activated lymphoid cells. Purified CD34-positive hematopoietic progenitors were uPAR negative, but expressed uPAR during differentiation in short-term liquid culture stimulated in vitro by recombinant interleukin (IL)-1, IL-3, IL-6, granulocyte-macrophage colony stimulating factor (CSF), granulocyte-CSF, and stem cell factor. Enhanced uPAR expression was also seen in HL-60 cells after induction of differentiation with dimethyl sulfoxide or 1 alpha,25-dihydroxyvitamin D3. In lymphomas and leukemias, the staining pattern was similar to that seen in the normal cells with labelling of monocytic and myeloid that seen in the normal cells with labelling of monocytic and myeloid malignancies, but not of the neoplastic cells in B-cell or T-cell lymphomas or Hodgkin's disease. In conclusion, uPAR is a differentiation marker for myeloid and monocytic cells, and may act to facilitate migration of these cells in normal and pathologic conditions by cell-associated plasminogen activation. Whether expression of uPAR in myeloid and monocytic malignancies relates to their growth and behavior will be an important topic for investigations in the future.

The binding of iron-saturated 125I-lactoferrin to human monocytes was studied at pH 7.4 and 37 degrees C. Monocytes in suspension bound 125I-lactoferrin by a reversible, saturable and specific binding indicating the presence of a receptor. The dissociation constant (KD) of the binding was estimated at about 4.5 x 10(-9) M and the number of receptors was about 1.6 x 10(6) per monocyte. The affinity of native lactoferrin (20% iron saturated) was only slightly below that of iron-saturated lactoferrin (KD about 7.9 x 10(-9) M). Human transferrin, horse cytochrome c and human immunoglobulin G were without inhibitory effect on the binding of 125I-lactoferrin. The majority of cell-bound 125I-lactoferrin was dissociable. The dissociation rate was not affected by addition of unlabelled lactoferrin to the dissociation medium. The binding of 125I-lactoferrin to adherent mononuclear blood cells showed an about 100-fold lower affinity (KD about 2.5 x 10(-7) M) than to cells in suspension, but the specificity of the binding was the same. These results are compatible with the idea that lactoferrin exerts a biological effect mediated by an interaction with cells of the monocyte/macrophage lineage.

The cellular urokinase‐type plasminogen‐activator (uPA) receptor (uPAR) is a glycolipid‐anchored membrane protein thought to be involved in pericellular proteolysis during cell migration and tumor invasion. In the present study, we have identified and characterized two soluble forms of uPAR which have retained their ligand‐binding capability. One variant was generated in vitro by treatment of intact normal cells with either a phosphatidylinositol‐specific phospholipase C (PLC) or endoproteinase Asp‐N. The other soluble uPAR variant was secreted in vivo from peripheral blood leukocytes affected by the stem‐cell disorder paroxysmal nocturnal hemoglobinuria (PNH), and was found in the plasma from these PNH patients as well as in the conditioned medium from cultured PNH leukocytes. Under normal conditions, we find no evidence for any shedding or secretion of a soluble uPA‐binding counterpart to human uPAR in plasma. Unlike normal leukocytes, the PNH‐affected cells do not express uPAR on the cell surface, although they do contain apparently normal levels of uPAR‐specific mRNA. The secreted uPAR derived from PNH cells has a mobility in SDS/PAGE that is slightly higher than that of uPAR solubilized by PtdIns‐specific PLC or detergent, but resembles that of a truncated, recombinant uPAR variant, which has its C‐terminus close to the proposed glycolipid‐attachment site, suggesting that the secreted protein has been proteolytically processed for glycolipid attachment. The presence in plasma from PNH patients of such a secreted, hydrophilic form of uPAR lends support to the hypothesis that the lesion underlying the PNH disorder resides either in glycolipid biosynthesis or in the function of an as‐yet‐unidentified trans‐amidating enzyme assumed to cleave and assemble the truncated uPAR with the preformed glycolipid moiety.

ABSTRACT. Neutrophil granulocyte chemotaxis and intraneutrophilic and plasma levels of lysozyme as well as the number of T and B lymphocytes and lymphocyte transformation in vitro on stimulation with mitogens and microbial antigens were studied in four groups of patients with diabetes mellitus (DM). Twelve patients with insulin‐dependent diabetes mellitus (IDDM) and ketoacidosis and 4 patients with non‐insulin‐dependent diabetes mellitus were studied at the time of diagnosis and before and after start of treatment. Ten patients with IDDM of less than 10 years' duration which had been difficult to regulate well and 10 patients with IDDM well regulated for more than 20 years were studied at their regular outpatient visits. Apart from a slight increase in plasma lysozyme in group 1 from the first to the second examination, we found no differences between diabetics and healthy control persons. It is concluded that if patients with DM are more susceptible to infections, it is probably caused by elements of neutrophil or lymphocyte function not examined in this study or by factors unrelated to immunity.

MBL levels have no discernible influence on the occurrence or course of infections in AML patients during the initial hospitalisation. The predominant immunodeficiency during this phase is the profound granulocytopenia, which also compromises important effector functions of MBL. The finding in most AML patients of elevated MBL concentrations on admission is most likely because of the role of MBL as an acute phase reactant.

The urokinase plasminogen activator (uPA) is a proteolytic enzyme which converts the proenzyme plasminogen to the active serine protease plasmin. A cell surface receptor for uPA (uPAR) is attached to the cell membrane by a glycosyl-phosphatidylinositol anchor. Binding of uPA to uPAR leads to an enhanced plasmin formation and thereby an amplification of pericellular proteolysis. We have shown previously that uPAR is expressed on normal blood monocytes and granulocytes, but is deficient on affected blood monocytes and granulocytes in patients with paroxysmal nocturnal haemoglobinuria (PNH), and that uPAR is present in plasma from these patients. In this study a newly established sensitive enzyme-linked immunosorbent assay (ELISA) has been applied for quantitation of uPAR in plasma. Unexpectedly, we found that uPAR is not only present in PNH plasma but also in plasma from healthy individuals. In 39 healthy individuals the mean plasma-uPAR value +/- SD was 31 +/- 15 pM, median 28 (range 11-108), and the corresponding value for six PNH patients was 116 +/- 67 pM, median 90 (range 61-228). The elevated uPAR-level in PNH patients was highly significant (Mann-Whitney test; P < 0.0001), and may possibly contribute to the propensity for thrombosis in PNH by inhibition of the fibrinolytic system. Binding of pro-uPA by uPAR in plasma may interfere with the appropriate binding of pro-uPA to cell-bound uPAR and therefore inhibit cell-associated plasmin generation and fibrinolysis. It is likely that the uPAR in normal plasma reflects the overall level of activity of the uPAR-mediated cell surface proteolysis. The present ELISA may be used for studies of uPAR levels in plasma from patients with conditions in which this activity might be increased, such as cancer and inflammatory disorders. Future studies will determine if uPAR in plasma is a parameter of clinical importance in these diseases.

Mycotoxins are fungi-born metabolites that can contaminate foods through mould-infected crops. They are a significant food/feed-safety issue across the globe and represent a substantial financial burden for the world economy. Moreover, with a changing climate and fungal biota, there is now much discussion about emerging mycotoxins that are measurable at significant levels in crops world-wide. Unfortunately, we still know very little about the bioavailability and toxic potentials of many of these less characterized mycotoxins, including the large family of enniatins. In this study, we present new occurrence data for enniatin A, A1, B, B1 and beauvericin in four Danish crops: oat, wheat, and barley from the 2010 harvest, and rye from 2011 harvest. The occurrence of the four enniatins were B > B1 > A1 > A. Enniatin B was detected in 100% of tested samples regardless of crop type. In addition to occurrence data, we report a proof-of-concept study using a human-relevant high-content hepatotoxicity, or "quadroprobe," assay to screen mycotoxins for their cytotoxic potential. The assay was sensitive for most cytotoxic compounds in the 0.009-100 µM range. Among eight tested mycotoxins (enniatin B, beauvericin, altenariol, deoxynivalenol, aflatoxin B1, andrastin A, citrinin, and penicillic acid), enniatin B and beauvericin showed significant cytotoxicity at a concentration lower than that for aflatoxin B1, which is the archetypal acute hepatotoxic and liver-carcinogenic mycotoxin. Hence, the quadroprobe hepatotoxicity assay may become a valuable assessment tool for toxicity assessment of mycotoxins in the future. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1658-1664, 2017.