The spectrum of ABCC8 mutations in Norwegian patients with congenital hyperinsulinism of infancy.

Potassium channels in the plasma membrane of the pancreatic beta cells are critical in maintaining glucose homeostasis by responding to ATP and coupling metabolic changes to insulin secretion. These channels consist of subunits denoted the sulfonylurea receptor SUR1 and the inwardly rectifying ion channel KIR6.2, which are encoded by the genes ABCC8 and KCNJ11, respectively. Activating mutations in the subunit genes can result in monogenic diabetes, whereas inactivating mutations are the most common cause of congenital hyperinsulinism of infancy (CHI). Twenty-six Norwegian probands with CHI were analyzed for alterations in ABCC8 and KCNJ11. Fifteen probands (58%) had mutations in the ABCC8 gene. Nine patients were homozygous or compound heterozygous for the mutations, indicating diffuse pancreatic disease. In five patients, heterozygous and paternally inherited mutations were found, suggesting focal disease. One patient had a de novo mutation likely to cause a milder, dominant form of CHI. Altogether, 16 different ABCC8 mutations (including the novel alterations W231R, C267X, IVS6-3C>G, I462V, Q917X and T1531A) were identified. The mutations IVS10+1G>T, R1493W and V21D occurred in five, three and two families, respectively. KCNJ11 mutations were not found in any patients. Based on our mutation screening, we estimate the minimum birth prevalence of ABCC8-CHI in Norway to 1:70,000 during the past decade. Our results considerably extend the knowledge of the molecular genetics behind CHI in Scandinavia.

The progenitor cell marker NG2/MPG promotes chemoresistance by activation of integrin-dependent PI3K/Akt signaling.

Chemoresistance represents a major problem in the treatment of many malignancies. Overcoming this obstacle will require improved understanding of the mechanisms responsible for this phenomenon. The progenitor cell marker NG2/melanoma proteoglycan (MPG) is aberrantly expressed by various tumors, but its role in cell death signaling and its potential as a therapeutic target are largely unexplored. We have assessed cytotoxic drug-induced cell death in glioblastoma spheroids from 15 patients, as well as in five cancer cell lines that differ with respect to NG2/MPG expression. The tumors were treated with doxorubicin, etoposide, carboplatin, temodal, cisplatin and tumor necrosis factor (TNF)alpha. High NG2/MPG expression correlated with multidrug resistance mediated by increased activation of alpha3beta1 integrin/PI3K signaling and their downstream targets, promoting cell survival. NG2/MPG knockdown with shRNAs incorporated into lentiviral vectors attenuated beta1 integrin signaling revealing potent antitumor effects and further sensitized neoplastic cells to cytotoxic treatment in vitro and in vivo. Thus, as a novel regulator of the antiapoptotic response, NG2/MPG may represent an effective therapeutic target in several cancer subtypes.

Molecular aspects of the mammalian cell cycle and cancer.

Cancer arises mainly from mutations in somatic cells. However, it is not the result of a single mutation, rather, it results from increasing genetic disarray accumulated over time. Tumorigenesis in humans is, therefore, a multistep and age-dependent process. The multiple mechanisms and multiple players involved in this process necessitate an understanding of the molecular mechanisms, in order to distinctively classify the tumor sample and to assess the risk and treatment of the disease.

Establishment of okadaic acid resistant cell clones using a cDNA expression library.

The mechanism whereby the universal apoptogen and serine/threonine phosphatase inhibitor okadaic acid (OA) kills cells, is still unclear. To create a novel tool for probing of OA action, fibroblasts were selected for OA-resistance after infection with a retroviral Jurkat T-cell cDNA expression library. Twenty-one clones were selected. Two of these (OAR1, OAR2) were studied in detail. OAR1 and 2 had each a retrovirally introduced short cDNA, corresponding to a human gene (oar1 and oar2, respectively) with unknown function. Reintroduction of oar1 or oar2 cDNA into wild-type cells reproduced the OA-resistant phenotype. OAR1 and 2 were cross-resistant to other phosphatase inhibitors (calyculin A, cantharidin), but not to staurosporine or microinjected Cytochrome c, thus, indicating a disturbance in a limited number of death pathways, upstream or independent of apaf-1/caspases-3/9. The action of OA involved caspase-dependent and caspase-independent components. Both components were less efficient in OAR1 and 2, than in wild-type cells. Subtle differences existed between OA-induced phosphoprotein patterns in wild-type cells, OAR1, and OAR2, indicating that a narrow selection of protein phosphorylation events had been targeted. We propose that the clones have defects in a hitherto non-elucidated signal pathway linking OA-induced protein phosphorylation to initiation of a death execution pathway provided with a caspase-dependent amplification loop. The novel OA-resistant cell clones will be used to elucidate the significance for apoptosis of oar1 and 2, their link to altered protein phosphorylation, and the potential link of the latter to initiation of apoptosis.

Cloning and characterization of an endo-beta-1,3(4)glucanase and an aspartic protease from Phaffia rhodozyma CBS 6938.

We describe the identification and expression cloning of two novel enzymes, a beta-glucanase and an aspartic protease, secreted from the basidiomycetous yeast Phaffia rhodozyma. A cDNA library from P. rhodozyma CBS 6938 was constructed, and full-length cDNA encoding an endo-1,3(4)-beta-glucanase (bg1) and an aspartic protease (pr1) were cloned by expression cloning in Saccharomyces cerevisiae W3124. The bg1 cDNA encodes a 424-residue precursor protein with a putative signal peptide. The pr1 cDNA encodes a 405-residue prepropolypeptide with an 81-residue leader peptide. The aspartic protease was purified and characterized. It has a molecular mass of 36 kDa, an isoelectric point of pH 7.5, a pH activity optimum at 4.0-6.0, and a temperature activity optimum around 40 degrees C. Both enzymes show only low sequence identity to other known enzymes.

Crystallization and preliminary X-ray studies of beta-1, 4-galactanase from Aspergillus aculeatus.

Recombinant beta-1,4-galactanase from Aspergillus aculeatus has been crystallized and characterized by X-ray diffraction. Crystals were obtained in hanging drops by vapour-diffusion under the conditions 30% PEG 400, 0.2 M CaCl2 and 0.1 M Na HEPES buffered to pH 7.5. The crystals diffract to 2.3 A resolution and belong to one of the orthorhombic space groups I222 or I212121. The unit-cell dimensions are a = 60.42, b = 88.94 and c = 129.08 A. With one molecule in the asymmetric unit, the corresponding solvent content is approximately 48%.

Comparison of expression systems in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha, Klyveromyces lactis, Schizosaccharomyces pombe and Yarrowia lipolytica. Cloning of two novel promoters from Yarrowia lipolytica.

We have compared expression systems based on autonomously replicating vectors in the yeasts Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis, Hansenula polymorpha and Yarrowia lipolytica in order to identify a more suitable host organism for use in the expression cloning method (Dalbøge and Heldt-Hansen, 1994) in which S. cerevisiae has traditionally been used. The capacity of the expression systems to secrete active forms of six fungal genes encoding the enzymes galactanase, lipase, polygalacturonase, xylanase and two cellulases was examined, as well as glycosylation pattern, plasmid stability and transformation frequency. All of the examined alternative hosts were able to secrete more active enzyme than S. cerevisiae but the relative expression capacity of the individual hosts varied significantly in a gene-dependent manner. One of the most attractive of the alternative host organisms, Y. lipolytica, yielded an increase which ranged from 4.5 times to more than two orders of magnitude. As the initially employed Y. lipolytica XPR2 promoter is unfit in the context of expression cloning, two novel promoter sequences for highly expressed genes present in only one copy on the genome were isolated. Based on sequence homology, the genes were identified as TEF, encoding translation elongation factor-1 alpha and RPS7, encoding ribosomal protein S7. Using the heterologous cellulase II (celII) and xylanase I (xylI) as reporter genes, the effect of the new promoters was measured in qualitative and quantitative assays. Based on the present tests of the new promoters. Y. lipolytica appears as a highly attractive alternative to S. cerevisiae as a host organism for expression cloning.

2D-PAGE examination of mRNA populations from Penicillium freii mutants deficient in xanthomegnin biosynthesis.

Penicillium freii (Lund and Frisvad 1994) mutants deficient in the synthesis of xanthomegnin were isolated. In vitro translated mRNA populations from selected radiation induced mutants and naturally occurring P. freii strains not able to produce xanthomegnin were examined by 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Specific translation products were absent in mutants and natural isolates unable to produce xanthomegnin metabolites. One mutant (TSM 73) did not produce several of these translation products, indicating that a mutation in a regulatory gene involved in xanthomegnin production had occurred.

Expression cloning, purification and characterization of a beta-1,4-galactanase from Aspergillus aculeatus.

Expression cloning has been used to isolate a cDNA encoding beta-1,4-galactanase from the filamentous fungus Aspergillus aculeatus. A cDNA library was prepared from mycelia, inserted in a yeast expression vector and transformed into Saccharomyces cerevisiae. Thirteen clones secreting galactanase activity were identified from a screening of approximately 2.5 x 10(4) yeast colonies. All clones expressed transcripts of the same galactanase gene. The cDNA was re-cloned in an Aspergillus expression vector and transformed into Aspergillus oryzae. The recombinant enzyme had a molecular weight of 44,000 Da, an isoelectric point of pH 2.85, a pH optimum of pH 4.0-4.5, and a temperature optimum of 45-65 degrees C, which is similar to values obtained for a beta-1,4-galactanase purified from A. aculeatus. The enzyme degraded unsubstituted galactan to galactose and galactobiose. The deduced primary sequence of the enzyme showed no apparent homology to any known enzyme, in accordance with this being the first reported beta-1,4-galactanase cDNA. However, the deduced amino-acid sequence of a Bacillus circulans DNA sequence containing an open reading frame (ORF) with no known function, showed 36% identity and 60% similarity to the galactanase amino-acid sequence.