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.

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.

Partial caudal duplication in a newborn associated with meningomyelocele and complex heart anomaly.

BACKGROUND: Caudal duplication is a spectrum of rare congenital anomalies with a possible heterogeneous pathogenesis including incomplete separation of monovular twins. METHODS: We report an autopsy case of a full-term infant with incomplete caudal duplication syndrome associated with multiple anomalies. RESULTS: These anomalies included a duplicated penis; double urinary bladder with an attenuated tunica muscularis; duplication of lower bowel with two ilia, appendices and colons; colonic hypogangliosis and left imperforated anus associated with rectourethral fistula. Other anomalies consisted of sacral meningomyelocele, sacral duplication with hypoplastic left sacrum and pelvic bones, muscle atrophy and hypoplasia of the left lower extremity, abnormal lobation of liver with stomach entrapment, omphalocele, and right atrial isomerism syndrome. The complex pattern of anomalies suggests the possibility that partial caudal duplication might be part of the spectrum of conjoined twinning.

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.

Fetal obstructive uropathy: patterns of renal pathology.

Fetal obstructive uropathy (FOU) is characterized by obstruction of the urethra, renal anomalies, ureterovesical dilatation, oligohydramnios, cryptorchidism, and abdominal muscle wall changes. The main objective of the present study was to better understand the relationship between FOU and renal pathology using a series of 15 male autopsy cases. A total of 11 cases with patent anus and 4 with imperforate anus were analyzed. Of the first group, most cases showed obstruction at the level of prostatic urethra. Seven cases showed obstruction at the level of the prostatic urethra and histologic study revealed scarring and partial or complete absence of the prostate, while in the remaining four cases the prostate was present. Of the cases with imperforate anus, two showed obstruction at the level of prostatic urethra, one showed posterior urethral valves, and one was obstructed at the proximal urethra. In all cases the kidneys showed mixed (dysplastic and cystic) changes with no significant differences between the two groups. An inverse correlation was observed between degree of renal dysplasia and gestational age, whereas the opposite was true for cystic changes. Distal and collecting tubules were more intensely immunoreactive to the anti-cytokeratin antibody when compared to proximal tubules. Moreover, anti-cytokeratin immunoreactivity was more prominent in tubules displaying cystic dilatation. DNA fragmentation analysis of renal tissue revealed a higher apoptosis of mesenchymal and tubular cells in the FOU cases, compared to gestational aged-matched controls. These results suggest that renal anomalies in FOU might be related to the gestational age at which the injury occurred and to the duration of the obstruction.

Traffic pattern of cystic fibrosis transmembrane regulator through the early exocytic pathway.

The pathway of transport of the cystic fibrosis transmembrane regulator (CFTR) through the early exocytic pathway has not been examined. In contrast to most membrane proteins that are concentrated during export from the ER and therefore readily detectable at elevated levels in pre-Golgi intermediates and Golgi compartments, wild-type CFTR could not be detected in these compartments using deconvolution immunofluorescence microscopy. To determine the basis for this unusual feature, we analyzed CFTR localization using quantitative immunoelectron microscopy (IEM). We found that wild-type CFTR is present in pre-Golgi compartments and peripheral tubular elements associated with the cis and trans faces of the Golgi stack, albeit at a concentration 2-fold lower than that found in the endoplasmic reticulum (ER). delta F508 CFTR, a mutant form that is not efficiently delivered to the cell surface and the most common mutation in cystic fibrosis, could also be detected at a reduced concentration in pre-Golgi intermediates and peripheral cis Golgi elements, but not in post-Golgi compartments. Our results suggest that the low level of wild-type CFTR in the Golgi region reflects a limiting step in selective recruitment by the ER export machinery, an event that is largely deficient in delta F508. We raise the possibility that novel modes of selective anterograde and retrograde traffic between the ER and the Golgi may serve to regulate CFTR function in the early secretory compartments.

A di-acidic (DXE) code directs concentration of cargo during export from the endoplasmic reticulum.

Efficient export of vesicular stomatitis virus glycoprotein (VSV-G), a type I transmembrane protein, from the endoplasmic reticulum requires a di-acidic code (DXE) located in the cytosolic carboxyl-terminal tail (Nishimura, N., and Balch, W. E. (1997) Science 277, 556-558). Mutation of the DXE code by mutation to AXA did not prevent VSV-G recruitment to pre-budding complexes formed in the presence of the activated form of the Sar1 and the Sec23/24 complex, components of the COPII budding machinery. However, the signal was required at a subsequent concentration step preceding vesicle fission. By using green fluorescence protein-tagged VSV-G to image movement in a single cell, we found that VSV-G lacking the DXE code fails to be concentrated into COPII vesicles. As a result, the normal 5-10-fold increase in the steady-state concentration of VSV-G in downstream pre-Golgi intermediates and Golgi compartments was lost. These results demonstrate for the first time that inactivation of the DXE signal uncouples early cargo selection steps from concentration into COPII vesicles. We propose that two sequential steps are required for efficient export from the endoplasmic reticulum.

Cargo can modulate COPII vesicle formation from the endoplasmic reticulum.

The COPII coat complex found on endoplasmic reticulum (ER)-derived vesicles plays a critical role in cargo selection. We now address the potential role of biosynthetic cargo in modulating COPII coat assembly and vesicle budding. The ER accumulation of vesicular stomatitis glycoprotein (VSV-G), a transmembrane protein, or the soluble PiZ variant of alpha1-antitrypsin, reduced levels of general COPII vesicle formation in vivo. Consistent with this result, conditions that prevent the export of VSV-G from the ER led to a significant inhibition of general COPII vesicle budding from ER microsomes and the export of an endogenous recycling protein p58 in vitro. In contrast, synchronized export of VSV-G stimulated COPII vesicle budding both in vivo and in vitro. Under conditions where VSV-G is retained in the ER, we find that it can to be recovered in pre-budding complexes containing COPII components. These results suggest that the export of biosynthetic cargo is integrated with ER functions involved in protein folding and oligomerization. The ability of biosynthetic cargo to prevent or enhance ER export suggests that interactions of cargo with the COPII machinery contribute to the formation of vesicles budding from the ER.

Neurotransmitter release from semi-intact synaptosomes.

We have developed a secretion assay composed of semi-intact synaptosomes from which transmitter release is optimally evoked by micromolar Ca2+ in the presence of cytosol. Transmitter release from this preparation reconstitutes known characteristics of regulated exocytosis and is accompanied by a marked decrease in synaptic vesicles. The assay is useful in characterizing the components known to be involved in transmitter release, and should also facilitate the identification of additional factors that are important for this process.

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.

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.

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.

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.

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 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.

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.

Regulation of constitutive exocytic transport by membrane receptors. A biochemical and morphometric study.

Biochemical and morphometric approaches were combined to examine whether constitutive secretory transport might be controlled by plasma membrane receptors, as this possibility would have significant physiological implications. Indeed, IgE receptor stimulation in rat basophilic leukemia cells potently increased the rate of transport of soluble pulse-labeled 35S-sulfated glycosaminoglycans from distal Golgi compartments to the cell surface. This effect was largely protein kinase C (PKC)-dependent. Direct activation of PKC also stimulated constitutive transport of glycosaminoglycans, as indicated by the use of agonistic and antagonistic PKC ligands. PKC ligands also had potent, but different, effects on the exocytic transport from distal Golgi compartments to the plasma membrane of a membrane-bound protein (vesicular stomatitis virus glycoprotein), which was slightly stimulated by activators and profoundly suppressed by inhibitors of PKC. Morphological analysis showed impressive changes of the organelles of the secretory pathway in response to IgE receptor stimulation and to direct PKC activation (enhanced number of buds and vesicles originating from the endoplasmic reticulum and Golgi and increase in surface and volume of Golgi compartments), suggestive of an overall activation of exocytic movements. These results show that rapid and large changes in constitutive transport fluxes and in the morphology of the exocytic apparatus can be induced by membrane receptors (as well as by direct PKC stimulation).