Autophagy occurs upstream or parallel to the apoptosome during histolytic cell death

Histolysis refers to a widespread disintegration of tissues that is morphologically distinct from apoptosis and often associated with the stimulation of autophagy. Here, we establish that a component of the apoptosome, and pivotal regulator of apoptosis, is also required for histolytic cell death. Using in vivo and ex vivo assays, we demonstrate a global apoptogenic requirement for dark, the fly ortholog of Apaf1, and show that a required focus of dark(-) organismal lethality maps to the central nervous system. We further demonstrate that the Dark protein itself is a caspase substrate and find that alterations of this cleavage site produced the first hypermorphic point mutation within the Apaf1/Ced-4 gene family. In a model of ;autophagic cell death', dark was essential for histolysis but dispensable for characteristic features of the autophagic program, indicating that the induction of autophagy occurs upstream or parallel to histolytic cell death. These results demonstrate that stimulation of autophagy per se is not a ;killing event' and, at the same time, establish that common effector pathways, regulated by the apoptosome, can underlie morphologically distinct forms of programmed cell death.     

Available lysine content in human milk: stability during manipulation prior to ingestion

Available lysine content is an indicator of protein quality and nutritional value of milk. Many studies have examined the effects of extraction, treatment and storage of human milk upon its components, though no references are found regarding the possible changes in milk quality as defined by its content in essential amino acids such as lysine. The present study investigates the available lysine content in human milk and the variations in lysine resulting from milk manipulation as follows: (a) Cold storage (refrigeration at 4 degrees C for 48 hours, and frozen for 15 days at -20 degrees C); (b) Thermal treatment under conditions of low (Holder)(63 degrees C/30 minutes) and high pasteurization (75 degrees C/15 seconds). The results obtained show a decrease in milk lysine concentration after storage in both refrigerated and frozen samples. Pasteurization causes a highly significant loss of available lysine. The lysine losses were greater on applying low pasteurization versus the more gentle conditions of high pasteurization. CONCLUSIONS: While manipulation through cold storage or thermal treatment does not affect the protein content of human milk, its protein quality is modified. When human milk must be subjected to hygienization, it is preferable to apply high temperature treatment (75 degrees C, 15 seconds) than habitual pasteurization (63 degrees C, 30 minutes).     

The effect of alpha-tocopherol on lipid peroxidation of microsomes and mitochondria from rat testis

The testis is a remarkably active metabolic organ; hence it is suitable not only for studies of lipid metabolism in the organ itself but also for the study of lipid peroxidation processes in general. The content of fatty acids in testis is high with a prevalence of polyunsaturated fatty acids (PUFA) which renders this tissue very susceptible to lipid peroxidation. Studies were carried out to evaluate the effect of alpha-tocopherol in vitro on ascorbate-Fe(++) lipid peroxidation of rat testis microsomes and mitochondria. Chemiluminescence and fatty acid composition were used as an index of the oxidative destruction of lipids. Special attention was paid to the changes produced on the highly PUFA [C20:4 n6] and [C22:5 n6]. Lipid peroxidation of testis microsomes or mitochondria induced a significant decrease of both fatty acids. Total chemiluminescence was similar in both kinds of organelles when the peroxidized without (control) and with ascorbate-Fe(++) (peroxidized) groups were compared. Arachidonic acid was protected more efficiently than docosapentaenoic acid at all alpha-tocopherol concentrations tested when rat testis microsomes or mitochondria were incubated with ascorbate-Fe(++). The maximal percentage of inhibition in both organelles was approximately 70%; corresponding to an alpha-tocopherol concentration between 1 and 0.25 mM. IC50 values from the inhibition of alpha-tocopherol on the chemiluminescence were higher in microsomes (0.144 mM) than mitochondria (0.078 mM). The protective effect observed by alpha-tocopherol in rat testis mitochondria was higher compared with microsomes, associated with the higher amount of [C20:4 n6]+[C22:5 n6] in microsomes that in mitochondria. It is proposed that the vulnerability to lipid peroxidation of rat testis microsomes and mitochondria is different because of the different proportion of PUFA in these organelles The peroxidizability index (PI) was positively correlated with the level of long chain fatty acids. The results demonstrated the protective effect of alpha-tocopherol on lipid peroxidation in microsomes and mitochondria from rat testis.     

On the nucleolar size and density in human early granulocytic progenitors, myeloblasts

Human myeloblasts were studied in bone marrow of patients suffering from chronic phase of chronic myeloid leukaemia to provide more information on the nucleolar diameter in these early granulocytic progenitors. These cells are a convenient model for such study since the number of myeloblasts in diagnostic bone marrow smears of investigated patients is larger than in not-leukemic persons because of the increased granulopoiesis. The nucleolar diameter was measured in myeloblasts after various cytochemical procedures such as methods for visualisation of RNA, DNA and proteins of AgNORs using digitized images and image processing. The results clearly demonstrated that values of the nucleolar diameter depended on the procedures used for visualising nucleoli. It seems to be also clear that a close relationship exists between the diameter of nucleoli and their number since the larger the number of nucleoli per cell the smaller their mean size. However, one of multiple nucleoli present in the nucleus is usually significantly larger. Moreover, the possibility exists that the variability of nucleolar diameter of leukemic myeloblasts and thus the heterogeneity of these cells might depend on various stages of the cell cycle as supported by nucleolar measurements on aging leukemic myeloblasts (K 562 cells) in vitro. Since the staining density of small and large nucleoli did not differ substantially after staining for RNA, it seems to be likely that the nucleolar size is directly related to the total RNA content in myeloblasts. In addition, karyometry combined with RNA cytochemistry still appears to be an useful tool to study nucleoli at the single cell level.     

A DNA vaccine encoding CCL4/MIP-1beta enhances myocarditis in experimental Trypanosoma cruzi infection in rats

Chagas' disease, caused by Trypanosoma cruzi, is a major cause of cardiovascular disease in Latin America. Exacerbated inflammation disproportional to parasite load characterizes chronic myocardial lesions in chagasic patients. Chemokines and their receptors are expected to account for the renewed inflammatory processes after the inoculation of the parasite, but their potential unique functions are far from being clear. Herein, we evaluated the effect of a DNA vaccine encoding CCL4/MIP-1beta, a CC-chemokine, in T. cruzi-elicited myocarditis in rats. Holtzman rats were given intramuscularly cardiotoxin and the CCL4/MIP-1beta DNA-containing plasmid (100microg) was delivered in this muscular site four times. Fourteen days after last immunization, animals were inoculated with a myotropical CL-Brener T. cruzi clone. Peak of parasitism was observed at day 15 after infection, preceding the peak of myocardial inflammation at day 20. Myocarditis was still intense at day 30, but the inflammatory infiltrates showed a more focal distribution. The expression of CCL2/MCP-1 and CCL4/MIP-1beta correlated closely with the kinetics of myocardial inflammation. The CCL4/MIP-1beta DNA vaccine induced an increase of the levels of the anti-CCL4/MIP-1beta observed in T. cruzi-infected animals. This was associated with an exacerbation of myocardial inflammation and fibrosis, although alterations in parasitemia and myocardial parasitism were not observed. Our data suggest that CCL4/MIP-1beta plays a role in preventing excessive inflammation and pathology rather than in controlling parasite replication.     

Monokine induced by interferon gamma and IFN-gamma response to a fusion protein of Mycobacterium tuberculosis ESAT-6 and CFP-10 in Brazilian tuberculosis patients

IFN-gamma responses to Mycobacterium tuberculosis antigens ESAT-6 and CFP-10 have been proposed as specific markers of M. tuberculosis infection. Monokine induced by gamma interferon (MIG/CXCL9) has been shown to be expressed by IFN-gamma stimulated mononuclear cells and to attract activated T-cells through the chemokine receptor CXCR3. Since MIG is induced early in the response to IFN-gamma, measuring MIG may provide an interesting marker to assess downstream IFN-gamma induced responses, in contrast to assays that mainly focus on quantifying production of IFN-gamma per se. We, therefore, investigated MIG and IFN-gamma responses to a fusion protein of ESAT-6 and CFP-10, and compared responses to the conserved mycobacterial antigen 85B (Ag85B) and purified protein derivative (PPD) of M. tuberculosis, in 29 BCG vaccine controls and 24 TB patients. IFN-gamma secreting cells were determined by ELISPOT, and MIG production was measured by ELISA and flow cytometry. Production of MIG in response to ESAT-6/CFP-10, Ag85B and PPD correlated overall with increased numbers of IFN-gamma secreting cells (r=0.55, P<0.0001). A significant increase was noted among patients compared to controls in the secretion of IFN-gamma and MIG following stimulation with ESAT-6/CFP-10 or PPD (P<0.05). Moreover, MIG intracellular expression was higher in TB patients compared to BCG vaccines (P<0.05) in response to ESAT-6/CFP-10 or PPD. We conclude that MIG production correlates significantly with enhanced T-cell IFN-gamma production induced by M. tuberculosis-specific antigens ESAT-6/CFP-10. These results point to MIG as a potential novel biomarker that may be helpful in assessing downstream responses induced by IFN-gamma in TB.     

Experimental transmission of Leishmania tropica to hyraxes (Procavia capensis) by the bite of Phlebotomus arabicus

The ability of the sand fly Phlebotomus (Adlerius) arabicus to transmit Leishmania tropica was studied experimentally using hyraxes (Procavia capensis), natural reservoir hosts of the parasite. Sand flies became infected with L. tropica after feeding on a lesion of needle-inoculated hyrax. Moreover, P. arabicus fed with L. tropica promastigotes transmitted the parasite to hyraxes by bite during a second bloodmeal. Although the animals remained asymptomatic after infective sand fly bite, they were PCR positive and infectious for naive sand flies. We have thus demonstrated cyclical transmission of L. tropica by P. arabicus in hyraxes. This confirms experimentally the vectorial competence of P. (Adlerius) arabicus, and demonstrates that asymptomatic reservoir hosts are infectious to appropriate vectors.     

Lagochilascaris minor third-stage larvae secrete metalloproteases with specificity for fibrinogen and native collagen

Dissemination of parasitic infections depends on migration through tissues and evasion from both hemostatic processes and immune responses from hosts. Metalloproteases play major roles in these mechanisms of pathogen-host interactions and, thus, are considered drug targets. In this study, we characterized metalloprotease activities in excretory/secretory (ES) products from third stage larvae (L3) of the ascarid Lagochilascaris minor, the causative agent of lagochilascariosis, which demonstrates an impressive migrating capacity across host tissues, including bone. Gel enzymography showed that ES products of L3 display two major gelatinolytic activities. Optimal proteolytic activity was found to occur at neutral/alkaline pH and was associated with two L. minor-secreted metalloproteases of 59 (SM59(Lm)) and 114kDa (SM114(Lm)). We next showed that ES products of L3 were able to hydrolyze fibrinogen and collagen I at neutral pH, but not BSA, in an extensive manner. Furthermore, we demonstrated that ES products of L3 mediate hydrolysis of the triple helical structure of collagen I fibers in mouse mesentery. These results suggest that ES proteases of L3 might facilitate both L. minor migration through host tissues by hydrolyzing collagens of the extracellular matrix and evasion from host hemostatic mechanisms by degrading fibrinogen.     

Hydrogen peroxide cleavage of the prion protein generates a fragment able to initiate polymerisation of full length prion protein

The prion protein is central to the disease pathogenesis of a variety of neurodegenerative diseases such as CJD. The protein is only able to initiate the disease process following post-translational modification. The main characteristic of this change is the ability of this altered isoform to polymerise. We wish to determine if altered cleavage of the protein could generate a protein fragment able to initiate polymerisation. During normal metabolic breakdown the protein is initially cleaved at a single site at around amino acid residue 111/112 in the mouse sequence. A second site before amino acid residue 90 has been postulated as an alternative cleavage point. We have provided evidence that hydrogen peroxide as low as 50 microM in the presence of copper, iron or manganese (but not nickel, magnesium or zinc) can cleave the recombinant protein near this site and requires a GXXH motif in the protein sequence. This reaction results in the production of 6 and 19 kDa fragments of the protein. This cleavage pattern occurs in prion proteins from different species (mouse, chicken and turtle) and is enhanced by modification of the octameric repeat region. The 19 kDa fragment produced by this reaction is protease sensitive. This fragment in a pure form caused the polymerisation of wild-type prion protein by a seeding mechanism. Therefore our results provide a possible mechanism by which altered cleavage of the prion protein could result in the kind of protein polymerisation associated with prion diseases.