Phagosome formation in Paramecium: roles of somatic and oral cilia and of solid particles as revealed by video microscopy

The roles of somatic and oral cilia and solid particles during digestive vacuole (DV) formation in Paramecium multimicronucleatum were investigated using video-enhanced and immunofluorescence microscopy. Membrane incorporation into DVs was found to increase linearly with increasing particle concentration. The rate of discoidal vesicle transport to the cytopharynx was not affected by particles, showing that particles are not required for membrane trafficking to the cytopharynx. However, the presence of particles leads to an increased membrane fusion between the cytopharyngeal membrane and the discoidal vesicles. When live cells lost their somatic cilia on the left-ventral side anterior to the oral region due to deciliation, membrane incorporation into newly formed DVs was strongly inhibited. Using video-enhanced microscopy, latex beads were seen to be loaded along the quadrulus on the dorsal surface of the buccal cavity, but few beads were seen next to the dorsal and ventral peniculi. Particle sequestration into a pre-formed nascent digestive vacuole (NDV) was studied in Triton X-100-permeabilized cells whose ciliary beating was reactivated by the addition of Mg-ATP. Both beat frequency and the percentage of cells containing bead-labeled NDV were dependent on the Mg-ATP concentration: the higher the beat frequency, the higher the percentage of cells with a bead-labeled NDV. These results suggest that ciliary beating is probably the only mechanism required for particle accumulation in the NDV, while a coordinated beating of the somatic cilia on the left-ventral side anterior to the oral region as well as the quadrulus moves particles into the NDV. The beating of the peniculi may somehow prevent the backward flow of particles out of the NDV.     

Sodium valproate inhibits glucose transport and exacerbates Glut1-deficiency in vitro

Anticonvulsant sodium valproate interferes with brain glucose metabolism. The mechanism underlying such metabolic disturbance is unclear. We tested the hypothesis that sodium valproate interferes with cellular glucose transport with a focus on Glut1 since glucose transport across the blood-brain barrier relies on this transporter. Cell types enriched with Glut1 expression including human erythrocytes, human skin fibroblasts, and rat astrocytes were used to study the effects of sodium valproate on glucose transport. Sodium valproate significantly inhibited Glut1 activity in normal and Glut1-deficient erythrocytes by 20%-30%, causing a corresponding reduction of Vmax of glucose transport. Similarly, in primary astrocytes as well as in normal and Glut1-deficient fibroblasts, sodium valproate inhibited glucose transport by 20%-40% (P < 0.05), accompanied by an up to 60% downregulation of GLUT1 mRNA expression (P < 0.05). In conclusion, sodium valproate inhibits glucose transport and exacerbates Glut1 deficiency in vitro. Our findings imply the importance of prudent use of sodium valproate for patients with compromised Glut1 function.     

Employing a superior BACE1 cleavage sequence to probe cellular APP processing

The involvement of beta-secretase (BACE1; beta-site APP-cleaving enzyme) in producing the beta-amyloid component of plaques found in the brains of Alzheimer's patients, has fueled a major research effort to characterize this protease. Here, we describe work toward understanding the substrate specificity of BACE1 that began by considering the natural APP substrate and its Swedish mutant, APPSw, and proceeded on to include oxidized insulin B chain and ubiquitin substrates. From these findings, and the study of additional synthetic peptides, we determined that a decapeptide derived from APP in which the P3-P2' sequence, ...VKM--DA..., was replaced by ...ISY--EV... (-- = beta site of cleavage), yielded a substrate that was cleaved by BACE1 seven times faster than the corresponding APPSw peptide, SEVNL--DAEFR. The expanded peptide, GLTNIKTEEISEISY--EVEFRWKK, was cleaved an additional seven times faster than its decapeptide counterpart (boldface), and provides a substrate allowing assay of BACE1 at picomolar concentrations. Several APP mutants reflecting these beta-site amino acid changes were prepared as the basis for cellular assays. The APPISYEV mutant proved to be a cellular substrate that was superior to APPSw. The assay based on APPISYEV is highly specific for measuring BACE1 activity in cells; its homolog, BACE2, barely cleaved APPISYEV at the beta-site. Insertion of the optimized ISY--EV motif at either the beta-site (Asp1) or beta'-site (Glu11) directs the rate of cellular processing of APP at these two accessible sites. Thus, we have identified optimal BACE1 substrates that will be useful to elucidate the cellular enzymatic actions of BACE1, and for design of inhibitors that might be of therapeutic benefit in Alzheimer's disease.     

Inferring the phylogeny of disjunct populations of the azure-winged magpie Cyanopica cyanus from mitochondrial control region sequences

The azure-winged magpie (AWM), Cyanopica cyanus, is found in Asia and Iberia. This remarkable disjunct distribution has been variously explained by either the sixteenth-century introduction of birds into Iberia from the Far East, or by the loss of individuals from the central part of their range as a result of Pleistocene glaciations. We have used the mitochondrial control region to undertake a molecular phylogenetic analysis of the AWM, with sequences examined from individuals collected from across the current distribution range and incorporating representatives of all currently defined subspecies. The Western birds are genetically distinct from their Asian congeners and their divergence is basal in the phylogenetic tree. This indicates that the AWM is native to Iberia and not the result of a recent introduction from Asia. In Asia, two major mitochondrial DNA lineages were identified. These correspond to an Inland Asia group and a Pacific Seaboard group, and are separated topographically by the Da Hingan Ling mountains and the Yellow Sea. Molecular clock estimates suggest that these divergences are associated with Pleistocene glaciations. Furthermore, our data do not support the current classification of the AWM into 10 subspecies, as defined based on morphology and geographical distribution.     

Phagosomal acidification in Paramecium: effects on lysosomal fusion

Phagosomes of paramecia and amoeba and endosomes of fibroblasts and other mammalian cells are acidified prior to lysosomal fusion. The question, whether the phagosomal acidification process in paramecia is required for phagosome-lysosome fusion, was studied using ionophores, weak bases, and cytochalasin B (CB) in combination with monoclonal antibodies, acid phosphatase (AcPase) cytochemistry, and lysosome morphometry. Digestive vacuoles (DVs) of known ages were treated and examined. In untreated cells, lysosome binding to the membrane of the acidified DV increased linearly with age and reached a maximum before lysosome-DV fusion. When the fusion of the acidosomes with the very young DVs was prevented by CB, causing a block in the normal vacuole-pH drop, lysosome binding to the DVs as well as the rate and extent of lysosome-DV fusion were all greatly reduced. These effects of CB were reversible. When present prior to acidification, three ionophores and two weak bases did not inhibit the acidosome-DV fusion but raised the phagosomal pH and reduced both the rates of DV acidification and of lysosome-DV fusion. However, when added after acidification but prior to lysosome-DV fusion, five of the six perturbants studied did not inhibit this fusion but prolonged the period when DVs remained AcPase positive. Lastly, lysosome-DV fusion rates were found to be related to acidification rates. We conclude that an inhibition in acidosome-DV fusion or a reduction in both the acidification rate and vacuolar-pH drop would inhibit lysosome-DV fusion.     

Cytofluorescence detection of adriamycin-mitochondria interactions in isolated, perfused rat heart

The major side-effect of the anthracycline anti-tumor drug adriamycin is a specific, dose-dependent cardiotoxicity. Impairment of mitochondrial function has been suggested to play an important role in this toxicity. The present study addresses the question as to whether direct drug-mitochondria interactions occur in the isolated, perfused rat heart. To this aim, cytofluorescence microscopy experiments were performed on thin cryosections. To demonstrate the applicability of this technique it is shown that adriamycin bound to isolated rat liver and heart mitochondria can be visualized through its characteristic fluorescence. Longitudinal sections from heart tissue perfused with 50 microM adriamycin display two distinct cellular sites of drug accumulation, i.e., nuclei which exhibit very bright fluorescence and, in addition, mitochondria which become significantly labeled with the drug. The mitochondrial localization of adriamycin is confirmed independently by quantification of the drug content of the mitochondrial fraction after cell fractionation. These results are discussed in the light of the potential role of adriamycin-nuclei versus adriamycin-mitochondria interactions in the deterioration of heart performance.     

Sequence analysis of the COOH terminus of the alpha-chain of the fourth component of human complement. Identification of the site of its extracellular cleavage

The predominant form of the fourth component of complement in human and murine plasma (C4p) has an Mr approximately 5,000 less than C4 secreted by hepatocytes or macrophages (C4s). Here we demonstrate that the difference in Mr results from excision of a peptide from the COOH terminus of the alpha-chain of C4s. The site of truncation of the alpha-chain of C4 in plasma was established by sequence analysis of the COOH-terminal cyanogen bromide fragment isolated by high performance liquid chromatography. Sequential Edman degradation, digestion with carboxypeptidase Y, analysis of amino acid composition, and partial sequence analysis of an overlapping tryptic peptide established the sequence of this peptide to be Glu-Ala-Asn-Glu-Asp-Tyr-Glu-Asp-Tyr-Glu-Tyr-Asp-Glu-Leu-Pro-Ala. Comparison of our results with reported sequences establishes the COOH-terminal alanine to be residue 748 in the alpha-chain. This identifies the site at which C4s is cleaved by an extracellular protease and suggests that the protease has an elastase-like activity.     

Atriopeptins: a family of potent biologically active peptides derived from mammalian atria

Extracts of rat atria are potent stimulators of sodium and urine excretion, and relax vascular and intestinal smooth muscle preparations. The structures of six biologically active peptides obtained from atrial extracts are reported here. Ion exchange chromatography of a low molecular weight fraction obtained by gel filtration of atrial extracts produced two natriuretic fractions: the first induced relaxation of intestinal smooth muscle strips only, whereas the second also relaxed vascular strips as well. From the first fraction four pure biologically active peptides obtained by reverse phase HPLC have been sequenced: the 21 amino acid peptide, designated atriopeptin I, and three homologs (des- ser1 -, des- ser1 -ser2-, and des- ser21 - atriopeptin I). From the second fraction two pure biologically active peptides were obtained, which had C-terminal extensions of atriopeptin I: atriopeptins II (23 amino acid residues) and III (24 residues), having respectively phe-arg and phe-arg-tyr C-termini. These results suggest that this family of six peptides, sharing the same 17 membered ring formed by an internal cystine disulfide, is derived from a common high molecular weight precursor.     

Monoclonal antibodies with a high degree of specificity for Listeria monocytogenes serotype 4b.

Strains of Listeria monocytogenes serotype 4b account for a large fraction of sporadic listeriosis cases, as well as all major food-borne epidemics attributed to this pathogen. We have identified a set of three monoclonal antibodies which showed a high degree of specificity for strains of L. monocytogenes serotype 4b. Two of these antibodies (c74.33 and c74.180, isotypes immunoglobulin M [IgM] and IgG3, respectively) recognized all serotype 4b strains, whereas antibody c74.22 (isotype IgG1) failed to recognize certain epidemic-associated strains. The corresponding antigens were located on the surface of the bacteria and were expressed following bacterial growth in different media and over a wide range of temperatures (4, 22, and 37 degrees C). Heating L. monocytogenes cells at 80,90, or 100 degrees C abolished reactivity for c74.22 but not for c74.33 MAb. These MAbs were negative for all of the non-Listeria strains tested, including representatives of several gram-negative and gram-positive species. The surface antigen recognized by c74.22 appeared to be associated with the ability of the bacteria to enter (invade) mammalian cells in culture.     

Multiple peptide synthesis using a single support (MPS3)

An automated multiple peptide synthesis method to synthesize, cleave, and purify several peptides simultaneously in a single batch has been developed. The technique is based on the synthesis of multiple peptides on a single solid phase support and is easily adapted to manual or to automated methods. The approach relies on coupling of amino acid mixtures to the resin and it has been found that DCC/HOBt gives the best coupling performance. Fast Atom Bombardment Mass Spectrometry (FAB-MS) was used to rapidly and efficiently identify the peptides in each synthetic mixture which significantly assisted the purification process by HPLC. The method has been successfully applied to the synthesis of magainin 2 and angiotensinogen peptides.