BACKGROUND The use of plasma-based resuscitation for trauma patients in hemorrhagic shock has been associated with a decrease in mortality. While some have proposed a beneficial effect through replacement of coagulation proteins, the putative mechanisms of protection afforded by plasma are unknown. We have previously shown in a cell culture model that plasma decreases endothelial cell permeability compared to crystalloid. The endothelial glycocalyx consists of proteoglycans and glycoproteins attached to a syndecan backbone, which together protect the underlying endothelium. We hypothesize that endothelial cell protection by plasma is due, in part, to its restoration of the endothelial glycocalyx and preservation of syndecan-1 after hemorrhagic shock. METHODS Rats were subjected to hemorrhagic shock to a mean arterial blood pressure of 30 mmHg for 90 minutes followed by resuscitation with either lactated Ringer’s solution (LR) or fresh plasma to a mean arterial blood pressure of 80 mm Hg and compared to shams or shock alone. After two hours, lungs were harvested for syndecan mRNA, immunostained with anti-syndecan-1, or stained with hematoxylin and eosin. To specifically examine the effect of plasma on the endothelium, small bowel mesentery was infused with a lanthanum-based solution, venules identified, and the glycocalyx visualized by electron microscopy. All data are presented as mean ±SEM. Results were analyzed by one-way ANOVA with Tukey post hoc tests. RESULTS Electron microscopy revealed degradation of the glycocalyx after hemorrhagic shock which was partially restored by plasma but not LR. Pulmonary syndecan-1 mRNA expression was higher in animals resuscitated with plasma (2.76 ± 0.03) compared to shock alone (1.39 ± 0.22) or LR (0.82 ± 0.03) and correlated with cell surface syndecan-1 immunostaining. Shock also resulted in significant lung injury by histopathology scoring (1.63 ± 0.26) which was mitigated by resuscitation with plasma (0.67 ± 0.17) but not LR (2.0 ± 0.25). CONCLUSION The protective effects of plasma may be due in part to its ability to restore the endothelial glycocalyx and preserve syndecan-1 after hemorrhagic shock.

The structure of Sindbis virus was determined by electron cryomicroscopy. The virion contains two icosahedral shells of viral-encoded proteins separated by a membrane bllayer of cellular origin. The three-dimensional structure of the Ice-embedded intact Sindbis virus, reconstructed from electron images, unambiguously shows that proteins in both shelas are arranged with the same icosahedral lattice of trianglation number T = 4. These studies also provide structural evidence ofcontact between the glycoprotein and the nucleocapsid protein across the membrane bilayer. The structural organization of Sindbis virus has profound implications for the morphogenesis of the alphaviruses. The observed interactions conflrm stoichiometric and speciflc protein associations that may be crucial for virlon stability and predict a mechanism for assembly.Membrane-containing viruses are assembled through the interaction of virus-encoded proteins with a host-cell membrane. For most enveloped viruses, this process involves two pathways. In one of these pathways, virus-encoded membrane proteins are cotranslationally integrated into membranes of the cell endoplasmic reticulum (1). These proteins are subsequently processed and delivered to a particular cell membrane (usually the plasma membrane) by a sequence of transport and processing events used by the cell in the maturation of its own membrane proteins. Virus membrane proteins, therefore, have proven to be important models for studying the processes involved in the maturation and in the targeting of cellular membrane proteins.While envelope proteins are transported to the plasma membrane, other viral proteins are translated in the cell cytoplasm and subsequently attach to the modified cellular membranes through the interaction of the cytoplasmic protein with cytoplasmic domains of the viral membrane glycoproteins (1). This association defines a critical step in the morphogenesis of the virus particle as it initiates and drives the process of envelopment of the core structure in the modified cellular membrane. The specific protein-protein interactions that occur during the fmal stages of assembly result in the production of a mature virus particle that maintains its structural integrity until its functional components interact with a potential host cell, and virus disassembly occurs.Sindbis virus, the prototype of the alphaviruses, achieves its mature structure in a distinctive fashion. Unlike many other enveloped viruses, the two-membrane glycoproteins of Sindbis virus (El and E2) are organized on the surface of the virus membrane as a precise triangulation number (T) = 4 icosahedron (2-5). The structure of this icosahedral lattice depends upon intramolecular disulfide bridges residing in the El glycoprotein (5, 6), and the integrity of the protein lattice has been demonstrated to determine the structure and stability of the membrane bilayer (6). Thus, whereas most enveloped viruses are described as including a membrane bilayer that contains virus-specified surface proteins an...

Carboxysomes are polyhedral bodies consisting of a proteinaceous shell filled with ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO). They are found in the cytoplasm of all cyanobacteria and some chemoautotrophic bacteria. Previous studies of Halothiobacillus neapolitanus and Nitrobacter agilis carboxysomes suggest that the structures are either icosahedral or dodecahedral. To determine the protein shell structure more definitively, purified H. neapolitanus carboxysomes were re-examined by cryo-electron tomography and scanning transmission electron microscopy (STEM). Due to the limited tilt angles in the electron microscope, the tomographic reconstructions are distorted. Corrections were made in the 3D orientation searching and averaging of the computationally extracted carboxysomes to minimize the missing data effects. It was found that H. neapolitanus carboxysomes vary widely in size and mass as shown by cryoelectron tomography and STEM mass measurements, respectively. We have aligned and averaged carboxysomes in several size classes from the 3D tomographic reconstruction by methods that are not model-biased. The averages reveal icosahedral symmetry of the shell, but not of the density inside it, for all the size classes.

We have studied the structure and characteristics of inclusion bodies formed by the enzyme beta-lactamase in the periplasmic space of Escherichia coli or in the cytoplasm, following expression of the protein without its signal sequence. Electron microscopy of highly purified protein aggregates using a novel sucrose gradient centrifugation procedure revealed striking morphological differences. Periplasmic inclusion bodies were essentially amorphous whereas the protein particles in the cytoplasm were highly regular. Depending on the cellular location, the inclusion bodies exhibited differences in protein composition even though they were formed by the expression of the same polypeptide chain. It was shown that the chaperonins GroEL and SecB are not incorporated into the inclusion bodies. Furthermore, the degree of solubilization of the inclusion bodies in the presence of denaturants and the sensitivity of the aggregated proteins to protease digestion indicated that the differences between cytoplasmic and periplasmic inclusion bodies extend to the conformation of the associated polypeptide chains.

Alphaviruses have the ability to induce cell-cell fusion after exposure to acid pH. This observation has served as an article of proof that these membrane-containing viruses infect cells by fusion of the virus membrane with a host cell membrane upon exposure to acid pH after incorporation into a cell endosome. We have investigated the requirements for the induction of virus-mediated, low pH-induced cell-cell fusion and cell-virus fusion. We have correlated the pH requirements for this process to structural changes they produce in the virus by electron cryo-microscopy. We found that exposure to acid pH was required to establish conditions for membrane fusion but that membrane fusion did not occur until return to neutral pH. Electron cryo-microscopy revealed dramatic changes in the structure of the virion as it was moved to acid pH and then returned to neutral pH. None of these treatments resulted in the disassembly of the virus protein icosahedral shell that is a requisite for the process of virus membrane-cell membrane fusion. The appearance of a prominent protruding structure upon exposure to acid pH and its disappearance upon return to neutral pH suggested that the production of a "pore"-like structure at the fivefold axis may facilitate cell penetration as has been proposed for polio (J. Virol. 74 (2000) 1342) and human rhino virus (Mol. Cell 10 (2002) 317). This transient structural change also provided an explanation for how membrane fusion occurs after return to neutral pH. Examination of virus-cell complexes at neutral pH supported the contention that infection occurs at the cell surface at neutral pH by the production of a virus structure that breaches the plasma membrane bilayer. These data suggest an alternative route of infection for Sindbis virus that occurs by a process that does not involve membrane fusion and does not require disassembly of the virus protein shell.