Active site histidine in pig liver aminolevulic acid dehydratase modified by diethylpyrocarbonate and protected by Zn2+ ions

1. The effect of diethylpyrocarbonate (DEP) (0.1-0.35 mM) on the purified pig liver amino-levulic acid dehydratase (ALA-D) containing 0.3 g-atoms Zn/subunit, under different pHs (6.0-7.5), temperature (0-18 degrees C) and time (0-60 min) was studied. 2. Three histidyl residues/subunit were modified by DEP (0.2 mM, pH 6.8), but activity was completely lost after the first one had reacted, indicating the presence of one histidine residue essential for ALA-D catalysis. Reactivation by treatment with hydroxylamine (0.7 mM, pH 7.0) confirmed that only histidine and no other nucleophile amino acids were directly involved in DEP inhibition. 3. Zn ions (0.5 mM) and the substrate ALA (5-10 mM) protected against DEP inactivation, protection was dependent on pH. 4. Sn, Se, Hg, Cd, Mn, Co and Pb (0.01-0.1 mM) did not significantly protect ALA-D against inactivation. 5. It is concluded that the substrate and Zn binding sites and the essential histidyl residues are in close proximity in the active center. It is proposed that in the catalytic synthesis of porphobilinogen from ALA, histidine groups have the specific role of transporting protons from the aqueous media to a hydrophobic active site.     

Primary structure of cytochrome c from the insect Ceratitis capitata.

The complete amino acid sequence of cytochrome c from the Dipterous Ceratitis capitata (serie Acalypterae) has been determined by combining automatic and manual methods of sequence analysis. No overlaps between positions 79 and 80, 86 and 87, 91 and 92 as well as between 99 and 100 were obtained. The alignment of these peptides was done by homology with other sequences of cytochromes c from insects already described. Comparison with the sequences of cytochromes c of other Diptera studied so far shows three changes (positions 50, 60 and 61, according to vertebrate cytochrome c numeration) from the Acalypteran Drosophila melanogaster and five changes (positions 9, 36, 50, 60 and 61) from that of the Calypteran Haematobia irritans.     

Enzymes for Pancreatic Islet Isolation Impact Chemokine-Production and Polarization of Insulin-Producing β-Cells with Reduced Functional Survival of Immunoisolated Rat Islet-Allografts as a Consequence

The primary aim of this study was to determine whether normal variations in enzyme-activities of collagenases applied for rat-islet isolation impact longevity of encapsulated islet grafts. Also we studied the functional and immunological properties of rat islets isolated with different enzyme preparations to determine whether this impacts these parameters. Rat-islets were isolated from the pancreas with two different collagenases with commonly accepted collagenase, neutral protease, and clostripain activities. Islets had a similar and acceptable glucose-induced insulin-release profile but a profound statistical significant difference in production of the chemokines IP-10 and Gro-α. The islets were studied with nanotomy which is an EM-based technology for unbiased study of ultrastructural features of islets such as cell-cell contacts, endocrine-cell condition, ER stress, mitochondrial conditions, and cell polarization. The islet-batch with higher chemokine-production had a lower amount of polarized insulin-producing β-cells. All islets had more intercellular spaces and less interconnected areas with tight cell-cell junctions when compared to islets in the pancreas. Islet-graft function was studied by implanting encapsulated and free islet grafts in rat recipients. Alginate-based encapsulated grafts isolated with the enzyme-lot inducing higher chemokine production and lower polarization survived for a two-fold shorter period of time. The lower survival-time of the encapsulated grafts was correlated with a higher influx of inflammatory cells at 7 days after implantation. Islets from the same two batches transplanted as free unencapsulated-graft, did not show any difference in survival or function in vivo. Lack of insight in factors contributing to the current lab-to-lab variation in longevity of encapsulated islet-grafts is considered to be a threat for clinical application. Our data suggest that seemingly minor variations in activity of enzymes applied for islet-isolation might contribute to longevity-variations of immunoisolated islet-grafts.     

A genetic interaction between hepatitis C virus NS4B and NS3 is important for RNA replication

Hepatitis C virus (HCV) nonstructural protein 4B (NS4B), a poorly characterized integral membrane protein, is thought to function as a scaffold for replication complex assembly; however, functional interactions with the other HCV nonstructural proteins within this complex have not been defined. We report that a Con1 chimeric subgenomic replicon containing the NS4B gene from the closely related H77 isolate is defective for RNA replication in a transient assay, suggesting that H77 NS4B is unable to productively interact with the Con1 replication machinery. The H77 NS4B sequences that proved detrimental for Con1 RNA replication resided in the predicted N- and C-terminal cytoplasmic domains as well as the central transmembrane region. Selection for Con1 derivatives that could utilize the entire H77 NS4B or hybrid Con1-H77 NS4B proteins yielded mutants containing single amino acid substitutions in NS3 and NS4A. The second-site mutations in NS3 partially restored the replication of Con1 chimeras containing the N-terminal or transmembrane domains of H77 NS4B. In contrast, the deleterious H77-specific sequences in the C terminus of NS4B, which mapped to a cluster of four amino acids, were completely suppressed by second-site substitutions in NS3. Collectively, these results provide the first evidence for a genetic interaction between NS4B and NS3 important for productive HCV RNA replication.