Getting into the Golgi.

The intermediate compartment residing between the endoplasmic reticulum (ER) and the Golgi is now recognized to be a dynamic structure that captures cargo released from the ER in COPII vesicular carriers and promotes recycling by COPI vesicular carriers. These and other findings now provide compelling evidence for the importance of this intermediate in balancing anterograde and retrograde flow through the early secretory pathway and in the formation and maintenance of the Golgi stack.

The organization of endoplasmic reticulum export complexes.

Export of cargo from the ER occurs through the formation of 60-70nm COPII-coated vesicular carriers. We have applied serial-thin sectioning and stereology to quantitatively characterize the three-dimensional organization of ER export sites in vivo and in vitro. We find that ER buds in vivo are nonrandomly distributed, being concentrated in regional foci we refer to as export complexes. The basic organization of an export complex can be divided into an active COPII-containing budding zone on a single ER cisterna, which is adjacent to budding zones found on distantly connected ER cisternae. These budding foci surround and face a central cluster of morphologically independent vesicular-tubular elements that contain COPI coats involved in retrograde transport. Vesicles within these export complexes contain concentrated cargo molecules. The structure of vesicular-tubular clusters in export complexes is particularly striking in replicas generated using a quick-freeze, deep-etch approach to visualize for the first time their three-dimensional organization and cargo composition. We conclude that budding from the ER through recruitment of COPII is confined to highly specialized export complexes that topologically restrict anterograde transport to regional foci to facilitate efficient coupling to retrograde recycling by COPI.

Sequential coupling between COPII and COPI vesicle coats in endoplasmic reticulum to Golgi transport.

COPI and COPII are vesicle coat complexes whose assembly is regulated by the ARF1 and Sar1 GTPases, respectively. We show that COPI and COPII coat complexes are recruited separately and independently to ER (COPII), pre-Golgi (COPI, COPII), and Golgi (COPI) membranes of mammalian cells. To address their individual roles in ER to Golgi transport, we used stage specific in vitro transport assays to synchronize movement of cargo to and from pre-Golgi intermediates, and GDP- and GTP-restricted forms of Sar1 and ARF1 proteins to control coat recruitment. We find that COPII is solely responsible for export from the ER, is lost rapidly following vesicle budding and mediates a vesicular step required for the build-up of pre-Golgi intermediates composed of clusters of vesicles and small tubular elements. COPI is recruited onto pre-Golgi intermediates where it initiates segregation of the anterograde transported protein vesicular stomatitis virus glycoprotein (VSV-G) from the retrograde transported protein p58, a protein which actively recycles between the ER and pre-Golgi intermediates. We propose that sequential coupling between COPII and COPI coats is essential to coordinate and direct bi-directional vesicular traffic between the ER and pre-Golgi intermediates involved in transport of protein to the Golgi complex.

Cargo selection by the COPII budding machinery during export from the ER.

Cargo is selectively exported from the ER in COPII vesicles. To analyze the role of COPII in selective transport from the ER, we have purified components of the mammalian COPII complex from rat liver cytosol and then analyzed their role in cargo selection and ER export. The purified mammalian Sec23-24 complex is composed of an 85-kD (Sec23) protein and a 120-kD (Sec24) protein. Although the Sec23-24 complex or the monomeric Sec23 subunit were found to be the minimal cytosolic components recruited to membranes after the activation of Sar1, the addition of the mammalian Sec13-31 complex is required to complete budding. To define possible protein interactions between cargo and coat components, we recruited either glutathione-S-transferase (GST)-tagged Sar1 or GST- Sec23 to ER microsomes. Subsequently, we solubilized and reisolated the tagged subunits using glutathione-Sepharose beads to probe for interactions with cargo. We find that activated Sar1 in combination with either Sec23 or the Sec23-24 complex is necessary and sufficient to recover with high efficiency the type 1 transmembrane cargo protein vesicular stomatitis virus glycoprotein in a detergent-soluble prebudding protein complex that excludes ER resident proteins. Supplementing these minimal cargo recruitment conditions with the mammalian Sec13-31 complex leads to export of the selected cargo into COPII vesicles. The ability of cargo to interact with a partial COPII coat demonstrates that these proteins initiate cargo sorting on the ER membrane before budding and establishes the role of GTPase-dependent coat recruitment in cargo selection.

AP-2-containing clathrin coats assemble on mature lysosomes.

Coat proteins appear to play a general role in intracellular protein trafficking by coordinating a membrane budding event with cargo selection. Here we show that the AP-2 adaptor, a clathrin-associated coat-protein complex that nucleates clathrin-coated vesicle formation at the cell surface, can also initiate the assembly of normal polyhedral clathrin coats on dense lysosomes under physiological conditions in vitro. Clathrin coat formation on lysosomes is temperature dependent, displays an absolute requirement for ATP, and occurs in both semi-intact cells and on purified lysosomes, suggesting that clathrin-coated vesicles might regulate retrograde membrane traffic out of the lysosomal compartment.

The Sar1 GTPase coordinates biosynthetic cargo selection with endoplasmic reticulum export site assembly.

Cargo selection and export from the endoplasmic reticulum is mediated by the COPII coat machinery that includes the small GTPase Sar1 and the Sec23/24 and Sec13/31 complexes. We have analyzed the sequential events regulated by purified Sar1 and COPII coat complexes during synchronized export of cargo from the ER in vitro. We find that activation of Sar1 alone, in the absence of other cytosolic components, leads to the formation of ER-derived tubular domains that resemble ER transitional elements that initiate cargo selection. These Sar1-generated tubular domains were shown to be transient, functional intermediates in ER to Golgi transport in vitro. By following cargo export in live cells, we show that ER export in vivo is also characterized by the formation of dynamic tubular structures. Our results demonstrate an unanticipated and novel role for Sar1 in linking cargo selection with ER morphogenesis through the generation of transitional tubular ER export sites.

Role of vesicle-associated syntaxin 5 in the assembly of pre-Golgi intermediates.

Syntaxins are thought to function during vesicular transport as receptors on the target membrane and to contribute to the specificity of membrane docking and fusion by interacting with vesicle-associated receptors. Here, syntaxin 5 (Syn5) was shown to be an integral component of endoplasmic reticulum-derived transport vesicles. This pool, but not the target, Golgi-associated Syn5 pool, was essential for the assembly of vesicular-tubular pre-Golgi intermediates and the delivery of cargo to the Golgi. The requirement for vesicle-associated Syn5 in transport suggests a reevaluation of the basis for operation of the early secretory pathway.

Differential regulation of exocytosis by calcium and CAPS in semi-intact synaptosomes.

Using a novel approach to measure exocytosis in vitro from semi-intact synaptosomes, we establish that the Ca2+-dependent release of glutamate requires cytosolic factors for mobilization from the reserve pool. The cytosolic activity for glutamate release was not satisfied by CAPS, a soluble component required for norepinephrine (NE) release. Moreover, the CAPS-independent glutamate release from synaptic vesicles (SVs) was 200-fold less sensitive to Ca2+ than that required for dense core vesicles (DCVs). The differential regulation of exocytosis by CAPS, Ca2+, and potential novel cytosolic factor(s) suggests that the docking and fusion machinery controlling DCVs has diverged from that regulating glutamate-containing SVs.

Total Artificial Heart Bridge to Transplantation for a Patient With Occult Intracardiac Malignancy: Case Report.

Malignancy is the leading cause of long-term morbidity and mortality after heart and other solid organ transplantation; therefore, great emphasis is placed on pre- and post-transplantation cancer screening. Even with meticulous screening during evaluation for heart transplant candidacy, an occult cancer may not be apparent. Here, we share the case of a 51-year-old man with refractory heart failure who underwent total artificial heart implantation as a bridge to transplantation with the surprise finding of an isolated deposit of metastatic carcinoid tumor nested within a left ventricular papillary muscle in his explanted heart. The primary ileal carcinoid tumor was identified and resected completely. After remaining cancer-free for 14 months, he was listed for heart transplantation and was transplanted 2 months later. He is currently 3.5 months out from heart transplantation and doing well, without evidence of recurring malignancy.

Atherosclerosis-prone branch regions in human aorta: microarchitecture and cell composition of intima.

The microarchitecture and cell composition of intima were studied at the macroscopically unaffected branch regions of human thoracic aorta using en face preparations, scanning and transmission electron microscopy, and immunohistochemistry. The endothelial lining showed a heterogeneous pattern and altered morphology including the areas of deendothelialization covered with platelets and dilated intercellular clefts. Leukocyte adhesion, accumulation of subendothelial macrophages and lymphocytes were characteristic of proximal and lateral zones, while the flow divider showed no significant accumulation of blood cells. Smooth muscle cells (SMCs) on the flow divider were elongated, in a contractile state, contacted side-by-side and did not contain lipid inclusions. In the lateral and proximal zones, intima appeared to be a network of stellate SMCs which were in contact through their processes. Most of the SMCs were in a synthetic state and many of them contained small lipid droplets. The number of procollagen I positive cells and the volume of extracellular components were most significant at the lateral zones rather than at the flow divider. We did not observe any difference in the rate of proliferation. Our results suggest that the intimal layer at the lateral and proximal zones has some distinct structural peculiarities, which provoke the development of initial atherosclerotic lesions at these sites. Such an intimal structure is probably caused by different flow patterns at these zone. However, only the totality of different morphological features exhibited in the area of altered vascular wall shear stress may be considered as a prerequisite for atherosclerotic lesions.

The morphology of valves and valve-like structures in the canine and feline thoracic duct.

The microanatomy and ultrastructure of the feline and canine thoracic duct and afferent lymphatics were studied by scanning and transmission electron microscopy. We found that the lymphatic vessels were always terminated by ostial valves of two shapes, crescent- and navicular-like, in a ratio of 4:1. Specific regulatory structures along the free edges of the valves, including marginal thickenings and buttresses, are described. The tissue and cellular organization of the valve endothelium showed distinct peculiarities, particularly in the orientation and shape of the cells and their microrelief. We found that valvular endothelial cells, especially ¿tip cells¿, which are situated in unfavourable lymphodynamic conditions, were characterized by an increased volume density of intermediate (probably vimentin-based) filaments, suggesting an accommodative mechanism involving such filaments.

Regeneration of the endothelium in the canine and feline thoracic duct.

Recent improvements in microsurgical techniques on lymphatic vessels facilitated the treatment of lymphoedema. But it is becoming clear that a successful treatment of lymphatic disease has to be based on knowledge of ongoing processes. Particularly, one of the important and unclear issue is cellular mechanisms of lymphatic regeneration. The regeneration of endothelial and the smooth muscle cells of the thoracic duct has been experimentally tested in vivo. The canine and feline thoracic duct was cryo-injured using 3 mm-based copper rod. Damaged endothelial cells remained attached to the substrate and lost unthrombogenecy within 48 hr. Adjacent EC restored the defect within 3 days by migration and proliferation. We observed that on the first day, the endothelial monolayer included some elongated multinuclear cells with blind silver lines whereas, on the third day, they were replaced by a population of smaller ECs with numerous mitoses. Organization of the monolayer was restituted within 7 days. The newly formed endothelium was similar to regenerating endothelium of arteries. In general, the clot that appeared at the zone of injury on the second day was dissolved by the third day. Occasionally, the dense polymorphic clot adhered to the wall and caused a delay in reendothelisation. Complete restitution of the tunica media which involved migration and proliferation mechanisms accompanied the endothelium regeneration.

Heterogeneity of smooth muscle cells in embryonic human aorta.

Cellular composition of aortas from 5- to 12-week and 18- to 28-week-old human embryos were investigated using immunocytochemistry, scanning and transmission electron microscopy. The aorta of the 5- to 12-week-old embryos consisted of three sublayers differing in cellular composition. The inner sublayer adjacent to the endothelium contained round and ovoid cells with synthetic phenotype. In the intermediate sublayer, spindle-like cells ultrastructurally similar to smooth muscle cells were found. Cells of the outer sublayer resembled fibroblasts or poorly differentiated mesenchymal cells. There were not definite morphological borders between sublayers. In the 18- to 28-week-old embryo aorta the intima was separated from media by internal elastic lamina. Intimal and innermost medial cells had predominately stellate shape and synthetic phenotype. The outer part of media contained spindle-like cells that had well developed contractile structures. Both the 5- to 12-week-old and the 18- to 28-week-old embryo aortic cells were positively stained for alpha-actin and myosin and negatively stained for macrophage antigens. Thus, the majority of embryo aortic cells appeared smooth muscle cells, however there was a regional difference in shape and synthetic state of these cells.

[The valvular apparatus and tissue organization of the endothelium of the thoracic duct]. / Klapannyi apparat i tkanevaia organizatsiia éndoteliia grudnogo protoka.

The structure of ostial valves and valves located along the thoracic duct and of its branches ostial valves and right lymphatic duct ostial valves were studied in 30 experimental outbred dogs and 46 cats. Cryodestruction of thoracic duct was performed in 28 outbred cats. 1, 3, 7 and 14 days later perfusive fixation with intercellular borders impregnation was carried out with simultaneous examination of intact regions of intravalvular segments, cisterna chyli and area of thoracic duct trunks connection with valvular surfaces. Tissue organization in ageing was studied using the intervalvular segment of old animals. Specimens were studied by means of scanning electron microscopy and film preparations of endothelium. Valves, located along the thoracic duct length are bicuspid formations, while ostial ones are falciform and cuneiform respectively in 80 and 20%. Endotheliocytes of cuspids are characterized with high content of microfilaments bundles in the cytoplasm and low content of microvesicles. Cells of the valvular free margin cross the cuspid edge and have adaptive changes preventing their desquamation: fusiform shape, long basal processes and bundles of microfilaments in the cytoplasm. Peculiar "pericyte-like" cells alike with myofibroblasts lie deep in the cuspid thickness close to the sinusal venous side. Fascicles of the duct smooth myocytes reach the base of the valve. Besides, in the ostial valve stroma there is elastic membrane, better displayed along the cuspid venous side. Increased polymorphism and changes of the endotheliocytes metric characteristics were demonstrated in the zones of turbulent lymph flow. Analysis of the newly formed endothelium tissue mosaics allows to reveal mechanisms of monolayer repair: spreading and migration of endotheliocytes on the first day, their proliferation within three days, desquamation of newly formed endotheliocytes and spreading of adjacent cells on later stages.

[The hemodynamic effect on the spatial organization of atherosclerotic lesions in the human aorta. Topical differences in the fatty streaks and in the lipofibrous and fibrous plaques]. / Vliianie gemodinamiki na prostranstvennuiu organizatsiiu ateroskleroticheskikh porazhenii aorty cheloveka. Topicheskie otlichiia v lipidnykh polosakh, lipofibroznykh i fibroznykh bliashkakh.

By means of light microscopy of whole-mounted membranous preparations and scanning electron microscopy differences of the three-dimensional organization in the fatty streaks, lipo-fibrous and fibrous plaques in proximal, distal and central parts were investigated. It was revealed that the central parts of the atherosclerotic lesions have a more mature character than the proximal and, especially, distal parts of the plaques. This phenomenon is accounted for by a progressive growth of the plaques along blood flow in distal direction due to the changes in hemodynamics.

[The hemodynamic effect on the spatial organization of atherosclerotic lesions in the human aorta. Plaques at the sites of arterial branching]. / Vliianie gemodinamiki na prostranstvennuiu organizatsiiu ateroskleroticheskikh porazhenii aorty chelovka. Bliashki v mestakh vetvleniia arterii.

By means of light microscopy of whole-mounted membranous preparations and scanning electron microscopy differences of the three-dimensional organization in the macroscopically intact and atherosclerotic human aortic intima in the sites of intercostal artery ostium were investigated. It was shown that the process of atherogenesis begins in the lateral areas and later involves the site of the entry. The flow divider does not contain lipids and resembles fibrous cap of the plaque. Such differences in the three-dimensional organization are accounted for by the local hemodynamic properties.

Surgical management and role of medical therapy in ruptured aneurysmal neurocysticercosis. A case report and review of the literature.

We describe the case of an 80-year-old Hispanic male with an acute subarachnoid hemorrhage (SAH) due to an inflammatory middle cerebral artery (MCA) aneurysm rupture. Two years prior to this episode, the patient had undergone a resection of a left intracranial neurocysticercosis lesion. A current CT, CTA and MRI showed significant SAH, a left MCA aneurysm and a cystic lesion compatible with neurocysticercosis. Intraoperatively, this aneurysm was found to be adjacent to a neurocysticercosis cyst, a diagnosis confirmed by surgical pathology. Only a few cases of subarachnoid hemorrhage due to an inflammatory brain aneurysm have been reported. Due to the associated higher incidence of intraoperative rupture and difficulty clipping, our paper highlights the importance of considering an inflammatory origin in patients with a history of neurocysticercosis and subarachnoid hemorrhage. This is the oldest patient on record reported for this diagnosis and surgery.

The tunica media of the thoracic duct contains a heterogeneous population of muscle cells.

The three-dimensional cyto-architecture and ultrastructure of cells composing the different regions of the tunica media of canine and feline thoracic ducts (ThD) were investigated by scanning and transmission electron microscopy. The tunica media of the intervalvular regions consists of 2-4 layers of circulatory oriented ribbon-like smooth muscle cells (SMCs) of the contractile phenotype. Spot-like membrane contacts similar to gap junctions were observed between SMCs. In the parietal part of the valve's sinus (PPVS), modified SMCs containing large lipid droplets were found. Occasionally, in the upper thoracic part of the ThD in the PPVS, we observed cardiomyocyte-like cells (CMLCs) arranged as circularly oriented bundles. Frequent interdigitation contacts were formed between SMCs and CMLCs, suggesting a possible involvement of the latter cell type in pacemakers activity in the ThD.