Here, we document for the first time the presence of the 26S proteasome and the ubiquitin pathway in a protozoan parasite that is in an early branch in the eukaryotic lineage. The 26S proteasome of Trypanosoma cruzi epimastigotes was identified as a high molecular weight complex (1400 kDa) with an ATP-dependent chymotrypsin-like activity against the substrate Suc-LLVY-Amc. This activity was inhibited by proteasome inhibitors and showed same electrophorectic migration pattern as yeast 26S proteasome in nondenaturating gels. About 30 proteins in a range of 25-110 kDa were detected in the purified T. cruzi 26S proteasome. Antibodies raised against the AAA family of ATPases from eukaryotic 26S proteasome and the T. cruzi 20S core specifically recognized components of T. cruzi 26S. To confirm the biological role of 26S in this primitive eukaryotic parasite, we analyzed the participation of the ubiquitin (Ub)-proteasome system in protein degradation during the time of parasite remodeling. Protein turnover in trypomastigotes was proteasome and ATP-dependent and was enhanced during the transformation of the parasites into amastigotes. If 20S proteasome activity is inhibited, ubiquitinated proteins accumulate in the parasites. As expected from the profound morphological changes that occur during transformation, cytoskeletal proteins associated with the flagellum are targets of the ubiquitin-proteasome pathway.
We used light and electron microscopic immunocytochemical methods to examine the structure of neuronal perikarya and processes containing cholecystokinin-like immunoreactivity (CCK-IR) in area CA1 of the rat hippocampus. The morphology of stained perikarya, their positions within all laminae, and the orientation of their dendrites indicate that CCK-IR is located in interneurons. These cells were seen in the electron microscope to have deeply folded nuclei and to receive both symmetric and asymmetric synaptic junctions on their cell somata and dendritic shafts. Their dendrites are essentially spine-free, but form bulges at the site of some asymmetric synaptic junctions. Axonal varicosities containing CCK-IR make symmetric synaptic junctions with cell somata and dendritic shafts of both pyramidal and non-pyramidal neurons. In addition, CCK-IR varicosities form symmetric junctions with unstained non-pyramidal neurons and with CCK-IR cells, suggesting either recurrent innervation of one cell on itself or interaction between interneurons. The presence of CCK-IR varicosities and synaptic junctions on pyramidal cells is in agreement with physiological data which indicate that CCK has a direct postsynaptic action. The observation of CCK-IR varicosities forming synaptic junctions on non-pyramidal cells suggests that CCK might also modify the response of interneurons.
Neural cell adhesion molecule (NCAM) is a plasma membrane glycoprotein that is thought to mediate adhesion between neuronal elements and play an important role in neural development. Although NCAM has been found in the adult brain, as well as the developing brain, little is known about its function or ultrastructural distribution. A monoclonal antibody which was directed against embryonic (E15-E17) mouse brain and identified 180 and 140 kDa molecular weight forms of NCAM was used to examine the immunohistochemical localization of NCAM in the rodent striatum. Light microscopic results in the adult mouse and rat showed that most NCAM180,140 immunoreactivity was localized to the relatively small population of medium and large-sized aspiny interneurons of the caudate nucleus and to the majority of neurons in the globus pallidus. Ultrastructural analysis revealed that reaction product in aspiny somata was present in discrete, closely spaced patches of cytoplasm along the inner face of the plasma membrane and was also prominent in somatic protrusions which were frequently apposed to synapsing axons. Distal aspiny and pallidal dendrites containing NCAM180,140 received numerous synaptic inputs. Within caudate neuropil NCAM180,140 was also present in spines with thin necks and small spine heads which were postsynaptic to unlabeled axon terminals and in preterminal (unmyelinated) axons and terminal boutons that issued from myelinated bundles and formed asymmetric synapses with unlabeled dendritic spines. This study provides the first evidence in adult brain that NCAM180,140 varies in extent and location within neurons. The heterogeneous distribution of NCAM180,140 in neurons of the basal ganglia may be related to a number of functions such as maintaining or modulating the density and distribution of synaptic inputs and the formation of new contacts on dendritic spines.
The initial, phase of membrane attack by complement is the interaction between C5b6, C7, and the cell membrane that leads to the insertion of C5b-7. Here we investigate the role of sialic acid residues in the assembly of C5b-7 intermediates on erythrocyte cell membranes. We find that C5b6 binds to glycophorin, whereas C5 or C6 does not bind, and desialylation of the glycophorin abolishes C5b6 binding. Complement lysis is inhibited by either masking glycophorin sialic acid with F~b fragments of an mAb, or by removal of the sialylated region of glycophorin by mild trypsinization. Gangliosides inhibit C5b-7 deposition when added to the aqueous phase. Asialogangliosides and synthetic gangliosides lacking the carboxylic acid residue have no inhibitory activity. We conclude that C5b6 binds to sialylated molecules on the erythrocyte surface. We propose a new model of membrane attack in which C5b6 initially binds to membranes via ionic forces. C7 then binds to C5b6, disrupting the ionic interaction and leading to the exposure of hydrophobic domains. Sialic acid is known to inhibit complement activation. Thus, these findings reveal a paradoxical role for sialic acid in complement attack; the presence of sialic acid inhibits the generation of C5b6, but once the membrane attack pathway is initiated, sialic acid enhances complement lysis.
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