Glycosylation enhances functional stability of the chemotactic cytokine CCL2

The human chemokine CCL2 gene was expressed in the yeast P.pastoris and gave rise to a mixture of differently glycosylated recombinant proteins. In comparison to non-glycosylated E.coli-derived CCL2, glycosylated yeast CCL2L was 4-20 times less active in a chemotactic assay in vitro. However, CCL2L could maintain full activity upon prolonged incubation at 37 degrees C, whereas the non-glycosylated chemokine readily lost activity. It could be hypothesized that glycosylation is a mechanism used by the organism to modulate CCL2 stability. The partial loss of specific activity due to glycosylation is balanced by the advantage of prolonging the effectiveness of chemokine. Thus, differential glycosylation allows one to obtain highly effective short-lived CCL2 or less-effective long-lived CCL2 and may thus represent a novel mechanism of adaptation to pathological versus physiological conditions.     

Use of expression constructs to dissect the functional domains of the CHS/beige protein: identification of multiple phenotypes

The Chediak-Higashi Syndrome (CHS) and the orthologous murine disorder beige are characterized at the cellular level by the presence of giant lysosomes. The CHS1/Beige protein is a 3787 amino acid protein of unknown function. To determine functional domains of the CHS1/Beige protein, we generated truncated constructs of the gene/protein. These truncated proteins were transiently expressed in Cos-7 or HeLa cells and their effect on membrane trafficking was examined. Beige is apparently a cytosolic protein, as are most transiently expressed truncated Beige constructs. Expression of the Beige construct FM (amino acids 1-2037) in wild-type cells led to enlarged lysosomes. Similarly, expression of a 5.5-kb region (amino acids 2035-3787) of the carboxyl terminal of Beige (22B) also resulted in enlarged lysosomes. Expression of FM solely affected lysosome size, whereas expression of 22B led to alterations in lysosome size, changes in the Golgi and eventually cell death. The two constructs could be used to further dissect phenotypes resulting from loss of the Beige protein. CHS or beigej fibroblasts show an absence of nuclear staining using a monoclonal antibody directed against phosphatidylinositol 4,5 bisphosphate [PtdIns(4,5) P2]. Transformation of beige j fibroblasts with a YAC containing the full-length Beige gene resulted in the normalization of lysosome size and nuclear PtdIns(4,5)P2 staining. Expression of the carboxyl dominant negative construct 22B led to loss of nuclear PtdIns(4,5)P2 staining. Expression of the FM dominant negative clone did not alter nuclear PtdIns(4,5) P2 localization. These results suggest that the Beige protein interacts with at least two different partners and that the Beige protein affects cellular events, such as nuclear PtdIns(4,5)P2 localization, in addition to lysosome size.     

Primary structure and reactive site of a novel wheat proteinase inhibitor of subtilisin and chymotrypsin

The proteinase inhibitor WSCI, active in inhibiting bacterial subtilisin and a number of animal chymotrypsins, was purified from endosperm of exaploid wheat (Triticum aestivum, c.v. San Pastore) by ion exchange chromatography and its complete amino acid sequence was established by automated Edman degradation. WSCI consists of a single polypeptide chain of 72 amino acid residues, has a molecular mass of 8126.3 Da and a pl of 5.8. The inhibition constants (Ki) for Bacillus licheniformis subtilisin and bovine pancreatic alpha-chymotrypsin are 3.92 x 10(-9) M and 7.24 x 10(-9) M, respectively. The inhibitor contains one methionine and of tryptophan residue and has a high content of essential amino acids (41 over a total of 72 residues), but no cysteines. The primary structure of WSCI shows high similarity with barley subtilisin-chymotrypsin isoinhibitors of the Cl-2 type and with maize subtilisinchymotrypsin inhibitor MPI. Significant degrees of similarity were also found between sequences of WSCI and of other members of the potato inhibitor I family of the serine proteinase inhibitors. The wheat inhibitor WSCI has a single reactive site (the peptide bond between methionyl-48 and glutamyl-49 residues) as identified by affinity chromatography and sequence analysis.     

Is a 3-month course of treatment with Peg-interferon effective in acute HCV hepatitis?

From February 2002, all the consecutive patients referring to the Department of Infectious Diseases, University of Turin, who were diagnosed as having acute HCV hepatitis were included in a prospective cohort study to evaluate if a 3-month course of Peg-Interferon alpha-2b (1.5 microg/kg once weekly) is effective to decrease the risk of progression to chronic disease. ALT and HCV-RNA measurements were scheduled at week 4 and 12 during treatment, and 24 weeks after the end of therapy. As of April 2003, ten patients were enrolled in the study. As to HCV genotype, seven patients had type 1 and 3 type non-1. At entry, median HCV-RNA level was 129500 (range: 3000-3100000 copies/mL) and six patients were symptomatic. Treatment was given within 20 days (range: 8-30) of the ALT peak. All patients completing 4 weeks (n = 9) and 12 weeks of treatment (n = 7) had undetectable HCV-RNA. Five patients who completed the 24-week follow-up after the end of treatment had a sustained viral response with ALT levels within normal range. Therapy was well tolerated in all patients. Even if our data are not definitive, our results show that once-weekly administration of Peg-interferon alfa-2b in patients with acute HCV infection may be an effective and convenient regimen.     

Protein kinase C zeta nuclear translocation mediates the occurrence of radioresistance in friend erythroleukemia cells

Friend erythroleukemia cells require high doses (15 Gy) of ionizing radiation to display a reduced rate of proliferation and an increased number of dead cells. Since ionizing radiation can activate several signaling pathways at the plasma membrane which can lead to the nuclear translocation of a number of proteins, we looked at the intranuclear signaling system activated by Protein Kinases C, being this family of enzymes involved in the regulation of cell growth and death. Our results show an early and dose-dependent increased activity of zeta and epsilon isoforms, although PKC zeta is the only isoform significantly active and translocated into the nuclear compartment upon low (1.5 Gy) and high (15 Gy) radiation doses. These observations are concomitant and consistent with an increase in the anti-apoptotic protein Bcl-2 level upon both radiation doses. Our results point at the involvement of the PKC pathway in the survival response to ionizing radiation of this peculiar cell line, offering PKC zeta for consideration as a possible target of pharmacological treatments aimed at amplifying the effect of such a genotoxic agent.     

Isolated durum wheat and potato cell mitochondria oxidize externally added NADH mostly via the malate/oxaloacetate shuttle with a rate that depends on the carrier-mediated transport

We investigated whether and how mitochondria from durum wheat (Triticum durum Desf.) and potato (Solanum tuberosum), isolated from etiolated shoots and a cell suspension culture, respectively, oxidize externally added NADH via the mitochondrial shuttles; in particular, we compared the shuttles and the external NADH dehydrogenase (NADH DHExt) with respect to their capacity to oxidize external NADH. We found that external NADH and NADPH can be oxidized via two separate DHExt, whereas under conditions in which the activities of NAD(P)H DHExt are largely prevented, NADH (but not NADPH) is oxidized in the presence of external malate (MAL) and MAL dehydrogenase, in a manner sensitive to several non-penetrant compounds according to the occurrence of the MAL/oxaloacetate (OAA) shuttle. In durum wheat mitochondria and potato cell mitochondria, the rate of NADH oxidation was limited by the rate of a novel carrier, the MAL/OAA antiporter, which is different from other carriers thought to transport OAA across the mitochondrial membrane. No NAD(P)H oxidation occurred arising from the MAL/Aspartate and the alpha-glycerophosphate/dihydroxyacetonphosphate shuttles. We determined the kinetic parameters of the enzymes and the antiporter involved in NADH oxidation, and, on the basis of a kinetic analysis, we showed that, at low physiological NADH concentrations, oxidation via the MAL/OAA shuttle occurred with a higher efficiency than that due to the NADH DHExt (about 100- and 10-fold at 1 microm NADH in durum wheat mitochondria and in potato cell mitochondria, respectively). The NADH DHExt contribution to NADH oxidation increased with increasing NADH concentration.     

Glutamate synthesis in barley roots: the role of the plastidic glucose-6-phosphate dehydrogenase

Evidence is provided for a close link between glutamate (Glu) synthesis and the production of reducing power by the oxidative pentose phosphate pathway (OPPP) in barley ( Hordeum vulgare L. var. Alfeo) root plastids. A rapid procedure for isolating organelles gave yields of plastids of over 30%, 60% of which were intact. The formation of Glu by intact plastids fed with glutamine and 2-oxoglutarate, both substrates of glutamate synthase (GOGAT), depends on glucose-6-phosphate (Glc-6-P) supply. The whole process exhibited an apparent K(m Glc-6-P) of 0.45 mM and is abolished by azaserine, a specific inhibitor of GOGAT; ATP caused a decrease in the rate of Glu formation. Glucose and other sugar phosphates were not as effective in supporting Glu synthesis with respect to Glc-6-P; only ribose-5-phosphate, an intermediate of OPPP, supported rates equivalent to Glc-6-P. Glucose-6-phosphate dehydrogenase (Glc6PDH) rapidly purified from root plastids showed an apparent K(m Glc-6-P) of 0.96 mM and an apparent K(m NADP)(+) of 9 micro M. The enzyme demonstrated high tolerance to NADPH, exhibiting a K(i) (NADPH) of 58.6 micro M and selectively reacted with antibodies against potato plastidic, but not chloroplastic, Glc6PDH isoform. The data support the hypothesis that plastidic OPPP is the main site of reducing power supply for GOGAT within the plastids, and suggest that the plastidic OPPP would be able to sustain Glu synthesis under high NADPH:NADP(+) ratios even if the plastidic Glc6PDH may not be functioning at its highest rates.     

Carcinogenesis in laboratory mice after low doses of ionizing radiation

Experimental data on the incidence of solid tumors from various long-term mouse studies performed at the Casaccia laboratories over several years were reconsidered, limiting the analysis to the results available for doses equal to or less than 17 cGy of neutrons and 32 cGy of X rays since these dose limits are reasonably close to the generally accepted low-dose levels for high- and low-LET radiation (i.e. D(high-LET) < 5 cGy and D(low-LET) < 20 cGy, respectively). The following long-term experiments with BC3F1 mice were reviewed: (a) females treated with single doses of 1.5 MeV neutrons or 250 kVp X rays, (b) males treated with fractionated doses of fission neutrons, and (c) mice of both sexes irradiated in utero 17.5 days post coitus with single doses of fission neutrons or X rays. An experiment with CBA mice of both sexes treated with single doses of fission neutrons was also included in this study. Analysis was done on animals at risk; thus all incidences of tumor-bearing animals were expressed as the percentage excess incidence with respect to the controls. Ovarian tumors and other solid neoplasms were considered. The percentage frequencies and mean survival times of tumor-free mice were also recalculated. The results indicate the existence of a region at low doses where the final incidence of solid neoplasms is indistinguishable from the background incidence. These data reinforce the idea that at low doses the effectiveness of ionizing radiation in inducing solid neoplasms in laboratory mice is very low.     

By binding SIRPalpha or calreticulin/CD91, lung collectins act as dual function surveillance molecules to suppress or enhance inflammation

Surfactant proteins A and D (SP-A and SP-D) are lung collectins composed of two regions, a globular head domain that binds PAMPs and a collagenous tail domain that initiates phagocytosis. We provide evidence that SP-A and SP-D act in a dual manner, to enhance or suppress inflammatory mediator production depending on binding orientation. SP-A and SP-D bind SIRPalpha through their globular heads to initiate a signaling pathway that blocks proinflammatory mediator production. In contrast, their collagenous tails stimulate proinflammatory mediator production through binding to calreticulin/CD91. Together a model is implied in which SP-A and SP-D help maintain a non/anti-inflammatory lung environment by stimulating SIRPalpha on resident cells through their globular heads. However, interaction of these heads with PAMPs on foreign organisms or damaged cells and presentation of the collagenous tails in an aggregated state to calreticulin/CD91, stimulates phagocytosis and proinflammatory responses.