Xenon tracers for cost effective laser induced fluorescence of alternative propellant Hall thrusters.

One of the limiting factors to developing plasma thrusters on alternative propellants is the cost associated with changing the diagnostic tools, which are often propellant-dependent. For laser induced fluorescence (LIF), which is typically used for ion velocity distribution measurements to determine ion trajectories and potential profiles, either new lasers need to be bought, which are tuned to the wavelength of the new element's excitation level, or a costly tunable laser is required. A method to use existing LIF setups designed for xenon on any propellant has been demonstrated on a Hall thruster operating on krypton. In the demonstration test, a small amount of xenon (0.01%-4%) was mixed with the main krypton propellant for use as a diagnostic tracer, and xenon ion velocities were measured while also monitoring changes in the mean discharge current and oscillations. High signal-to-noise ratios in LIF data acquired along the channel centerline were obtained with tracer gas fractions ≤1% that negligibly affected the thruster operation. These results and comparison of the emission spectra of xenon and other common propellants suggest that the tracer LIF method should be broadly applicable to LIF measurements in Hall thrusters operating on alternative propellants.

Energetics of side chain packing in staphylococcal nuclease assessed by exchange of valines, isoleucines, and leucines.

To examine the importance of side chain packing to protein stability, each of the 11 leucines in staphylococcal nuclease was substituted with isoleucine and valine. The nine valines were substituted with leucine and isoleucine, while the five isoleucines, previously substituted with valine, were substituted with leucine and methionine. These substitutions conserve the hydrophobic character of these side chains but alter side chain geometry and, in some cases, size. In addition, eight threonine residues, previously substituted with valine, were substituted with isoleucine to test the importance of packing at sites normally not occupied by a hydrophobic residue. The stabilities of these 58 mutant proteins were measured by guanidine hydrochloride denaturation. To the best of our knowledge, this is the largest library of single packing mutants yet characterized. As expected, repacking stability effects are tied to the degree of side chain burial. The average energetic cost of moving a single buried methyl group was 0.9 kcal/mol, albeit with a standard deviation of 0.8 kcal/mol. This average is actually slightly greater than the value of 0.7-0.8 kcal/mol estimated for the hydrophobic transfer energy of a methylene from octanol to water. These results appear to indicate that van der Waals interactions gained from optimal packing are at least as important in stabilizing the native state of proteins as hydrophobic transfer effects.

Enzymatic autocatalysis of botulinum A neurotoxin light chain.

Highly purified recombinant zinc-endopeptidase light chain of the botulinum neurotoxin serotype A underwent autocatalytic proteolytic processing and fragmentation. In the absence of added zinc, initially 10-28 residues were cleaved from the C-terminal end of the 448-residue protein followed by the appearance of an SDS-stable dimer and finally fragmentation near the middle of the molecule. In the presence of added zinc, the rate of fragmentation was accelerated but the specificity of the cleavable bond changed, suggesting a structural role for zinc in the light chain. The C-terminal proteolytic processing was reduced, and fragmentation near the middle of the molecule was prevented by adding the metal chelator TPEN to the light chain. Similarly, adding a competitive peptide inhibitor (CRATKML) of the light-chain catalytic activity also greatly reduced the proteolysis. With these results, for the first time, we provide clear evidence that the loss of C-terminal peptides and fragmentation of the light chain are enzymatic and autocatalytic. By isolating both the large and small peptides, we sequenced them by Edman degradation and ESIMS-MS, and mapped the sites of proteolysis. We also found that proteolysis occurred at F266-G267, F419-T420, F423-E424, R432-G433, and C430-V431 bonds in addition to the previously reported Y250-Y251 and K438-T439 bonds.

Development of vaccines for prevention of botulism.

Botulism is a potentially lethal disease caused by one of seven homologous neurotoxic proteins usually produced by the bacterium, Clostridium botulinum. This neuromuscular disorder occurs through an exquisite series of molecular events, ultimately ending with the arrest of acetylcholine release and hence, flaccid paralysis. The development of vaccines that protect against botulism dates back to the 1940s. Currently, a pentavalent vaccine that protects against BoNT serotypes A-E and a separate monovalent vaccine that protects against BoNT serotype F are available as Investigational New Drugs. However, due to the numerous shortcomings associated with the toxoid vaccines, several groups have efforts towards developing next-generation vaccines. Identifying a synthetic peptide that harbors a neutralizing epitope is one approach to a BoNT vaccine, while another employs the use of a Venezuelan equine encephalitis virus replicon vector to produce protective antigens in vivo against BoNT. The strategy used in our laboratory is to design synthetic genes encoding non-toxic, carboxy-terminal fragments of the C. botulinum neurotoxins (rBoNT(H(C))). The gene products are expressed in the yeast, Pichia pastoris, and purified to greater than 98% with yields typically ranging from 200-500 mg per kg of wet cells. Protective immunity to the purified products against high-level challenges of neurotoxin is elicited in mice and in non-human primates. A pre-Investigational New Drug meeting was held with the Food and Drug Administration, and the next milestone for the vaccine candidates will be clinical trials.

Fermentation, purification, and efficacy of a recombinant vaccine candidate against botulinum neurotoxin type F from Pichia pastoris.

A recombinant vaccine candidate was developed that protected mice against botulinum neurotoxin serotype F (BoNTF) intoxication. A synthetic gene encoding BoNTF fragment C (rBoNTF(H(c))) was designed, constructed, and inserted into a plasmid for expression in the yeast Pichia pastoris. A total cell protein content of 2.9 g was obtained per liter of fermentation broth. Recombinant rBoNTF(H(c)) was purified from the soluble yeast extract in two chromatographic steps. The process employed Mono S cation exchange chemistry followed by Alkyl-Superose hydrophobic interaction chromatography, producing material judged to be greater than 98% pure by SDS-PAGE. The recovery of purified product from cell extract was estimated to be greater than 42%, with a yield of 140 mg/kg of cell paste. rBoNTF(H(c)) was also purified from the insoluble fraction of the yeast cell lysate. Because the fragment C in the pellet was 35% of the total insoluble protein, only a Mono S cation exchange chromatography step was necessary to achieve a purity greater than 98%. Mice that received three injections of 0.2 microgram of purified soluble rBoNTF(H(c)) were completely protected when challenged with 1000 mouse ip LD(50) of BoNTF toxin. Similarly, three doses of 1 microgram of purified resolubilized rBoNTF(H(c)) completely protected mice from a challenge of 5000 mouse ip LD(50) of BoNTF toxin. Individual serum antibody ELISA titers of mice injected with soluble rBoNTF(H(c)) correlated with survival as all 34 mice with ELISA titers of 100 or greater survived toxin challenge. The work presented here demonstrates that purified rBoNTF(H(c)) is able to protect against a high challenge dose of neurotoxin.

Laparoscopic transcystic management of choledocholithiasis.

Our objective was to review our community hospital experience with laparoscopic management of choledocholithiasis from 1991 to 1997. We performed a retrospective review of all case records of patients with choledocholithiasis managed surgically at St. Francis Hospital during the study period. Data regarding the history, presentation, investigations, operative details, and follow-up were recorded. Procedures were performed by multiple attending surgeons supervising surgical residents. All common bile duct explorations (CBDEs) were performed by a transcystic approach and followed routine cholangiography. In most cases, cystic duct dilatation over a guide wire was followed by transcystic CBDE with choledochoscopy. Stone extraction was accomplished through a combination of flushing, basket manipulation, fragmentation, retrieval, or advancement of stones through the ampulla. Data were analyzed using SPSS computer software, and P < 0.05 was considered statistically significant. During the period of study there were 1053 laparoscopic cholecystectomies with and without cholangiography and 100 total CBDE performed. Of these, 54/100 had an attempt at laparoscopic CBDE. There were 39 females and 15 males, with a median age of 52 years (range 14-88). Presentation included acute cholecystitis or biliary colic (63%), gallstone pancreatitis (20%), and jaundice or cholangitis (17%). Successful laparoscopic stone removal was achieved in 36 of 54 (67%) cases. Eighteen of the remainder (33%) were converted to an open procedure. Size, number, position of stones, technical difficulties in accessing the common bile duct, and patient factors contributed to open conversion. The rate of successful laparoscopic CBDE improved for each individual surgeon from an average of 22 per cent in the first half of the study period (1991-1994) to 87 per cent in the second half (1995-1997). There was no operative mortality. Significant morbidity in the laparoscopic group included one retained stone and two cases of postoperative pancreatitis. There were three false negative preoperative endoscopic retrograde cholangiopancreatography examinations. Multivariate analysis showed that experience of the individual surgeon was the only significant factor predicting successful laparoscopic CBDE. Low initial success rate in the early phase of the study period improved dramatically to reach an overall success rate of 87 per cent in the second half. Laparoscopic management of common bile duct stones is possible in a community setting with a high success rate and minimal morbidity. It precludes excessive use of endoscopic retrograde cholangiopancreatography with its own set of complications but is associated with a significant learning curve. It is currently our preferred therapeutic approach for choledocholithiasis discovered pre- or intraoperatively.

Purification, potency, and efficacy of the botulinum neurotoxin type A binding domain from Pichia pastoris as a recombinant vaccine candidate.

Recombinant botulinum neurotoxin serotype A binding domain [BoNT/A(Hc)], expressed in Pichia pastoris, was developed as a vaccine candidate for preventing botulinum neurotoxin type A (BoNT/A) intoxication. After fermentation and cell disruption, BoNT/A(Hc) was purified by using a three-step chromatographic process consisting of expanded-bed chromatography, Mono S cation-exchange chromatography, and hydrophobic interaction chromatography. Two pools of immunogenic product were separated on the Mono S column and processed individually. Both products were more than 95% pure and indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). Each protein was assayed for potency in mice at immunogen doses ranging from 2.4 ng to 10 microg, followed by challenge with 1,000 mouse intraperitoneal 50% lethal doses (i.p. LD50) of BoNT/A. The calculated 50% effective dose for both peaks was approximately 0.1 microg/mouse. Peak 1 was evaluated further in a mouse efficacy assay. Mice were injected either once, twice, or three times at five different doses and subsequently challenged with 100,000 mouse i.p. LD50 of BoNT/A. In general, multiple injections protected better than one, with complete or nearly complete protection realized at doses of >/=0.5 microg/mouse. Serum neutralization and ELISA titers were also determined. Tellingly, 82 of 83 mice with antibody titers of >/=1, 600, as measured by ELISA, survived, but only 6 of 42 mice with titers of

Mustard gas crosslinking of proteins through preferential alkylation of cysteines.

Mustard gas, bis(2-chloroethyl)sulfide, treatment of proteins is shown to generate significant amounts of covalently crosslinked protein dimers. This is due to the preferential alkylation of cysteine residues. Crosslinking does not occur in the model protein staphylococcal nuclease, which has no cysteine residues. Treatment of cysteine-containing mutants of staphylococcal nuclease with this chemical warfare agent did result in crosslinking. However, these dimers are slowly cleaved back to monomers by an unknown mechanism. The alkylation and crosslinking of cysteine-containing proteins by mustard gas may contribute to its toxicity.

Chemically crosslinked protein dimers: stability and denaturation effects.

Nine single substitution cysteine mutants of staphylococcal nuclease (nuclease) were preferentially crosslinked at the introduced cysteine residues using three different bifunctional crosslinking reagents; 1,6-bismaleimidohexane (BMH), 1,3-dibromo-2-propanol (DBP), and the chemical warfare agent, mustard gas (bis(2-chloroethyl)sulfide; mustard). BMH and mustard gas are highly specific reagents for cysteine residues, whereas DBP is not as specific. Guanidine hydrochloride (GuHCl) denaturations of the resulting dimeric proteins exhibited biphasic unfolding behavior that did not fit the two-state model of unfolding. The monofunctional reagent, epsilon-maleimidocaproic acid (MCA), was used as a control for the effects of alkylation. Proteins modified with MCA unfolded normally, showing that this unusual unfolding behavior is due to crosslinking. The data obtained from these crosslinked dimers was fitted to a three-state thermodynamic model of two successive transitions in which the individual subunits cooperatively unfold. These two unfolding transitions were very different from the unfolding of the monomeric protein. These differences in unfolding behavior can be attributed in large part to changes in the denatured state. In addition to GuHCl titrations, the crosslinked dimers were also thermally unfolded. In contrast to the GuHCl denaturations, analysis of this data fit a two-state model well, but with greatly elevated van't Hoff enthalpies in many cases. However, clear correlations between the thermal and GuHCl denaturations exist, and the differences in thermal unfolding can be rationalized by postulating interactions of the denatured crosslinked proteins.

Energetic contribution of side chain hydrogen bonding to the stability of staphylococcal nuclease.

Hydrogen bonds are a ubiquitous feature of protein structures, yet there is great uncertainty about the energetic contribution of hydrogen bonding to protein stability. This study addresses this question by making a series of single substitution mutations in the model protein staphylococcal nuclease. These mutants have had a residue capable of participating in hydrogen bonding either removed or introduced. The variants we have investigated are as follows: nine valines substituted with threonine and serine; eight threonines converted to valine, serine, and cysteine; and seven tyrosines replaced by phenylalanine and leucine. The stabilities of these 56 mutant proteins were determined by titration with guanidine hydrochloride using fluorescence as a probe of structure. In general, it was found that the stability effects of removing a hydrogen bonding residue and replacing it with a nonbonding residue were relatively small. This was true even in the case of buried residues participating in hydrogen bonds, where the substituted residue leaves an unfulfilled hydrogen bond in the hydrophobic core. In contrast, introducing a hydrogen bonding residue in place of a nonbonding residue was generally more costly energetically. A wide variability in the cost of burying a hydroxyl was observed, but this does not seem to be due to differences in hydrogen bonding. The overall energetic contribution of various wild-type hydrogen bonding interactions was evaluated as being favorable. A range of energies from approximately 1.5 to 4.0 kcal/mol was estimated for the contribution of these interactions to the stability of the native state.

Effects of completion angiography on the outcome of carotid endarterectomy.

The carotid endarterectomy results of a single surgeon were analyzed over an 8-year period to determine how routine completion angiography affected endarterectomy outcome. Completion angiography was performed in 80% of cases. A total of 145 patients (86%) were symptomatic while 23 (14%) were symptom-free. Of 131 completion angiographies performed, 94 were interpreted as normal and 37 abnormal. The arteries were reopened in 23 patients with abnormal results. In the 14 patients with abnormal angiograms who were not reopened, two showed no flow into the internal carotid artery and the vessels were ligated. The remaining 12 patients had abnormalities in the external carotid artery or normal Doppler signals along the common carotid artery and internal carotid artery segments. Among the 37 patients with abnormal angiograms, arteries were re-explored in four; there were no neurologic complications. In the entire group, there were three mortalities, one stroke, one transient ischemic attack, two wound complications, four myocardial infarctions, two occurrences of cerebral edema, two pneumonias and nine transient local nerve injuries. Completion angiography has allowed an improved technique while also enhancing the ability to identify technical problems during surgery without increasing morbidity. These results support the use of intraoperative completion angiography to evaluate carotid endarterectomy sites.

Instrumentation for automated determination of protein stability.

A new instrument system has been developed that automatically carries out solvent and thermal denaturations of proteins using fluorescence as a probe of structure. This instrument also automatically performs pH titrations and can make kinetic measurements on the time scale of seconds. The design philosophy and implementation are described. The prototype instrument was subjected to extensive testing. The instrument can very reproducibly and accurately collect data that allow the calculation of the thermodynamics of protein denaturation. This data collection can proceed without human intervention after acquisition start until the data are saved.

Does the asymptomatic limb harbor deep venous thrombosis?

Deep venous thrombosis (DVT) is a great masquerader that cannot be reliably predicted by a patient's symptoms, history, or risk factors. Bilateral lower extremity duplex ultrasonography scans were made of 2,511 patients and analyzed to identify, if possible, a population in which a unilateral study would be appropriate. A total of 1,086 (43%) patients were found to have deep venous thrombosis--742 (30%) unilateral and 344 (14%) bilateral. Of the patients with DVT for whom side-of-symptom information was recorded, 64% had symptoms referable to the involved extremity and 36% had symptoms referable to the contralateral extremity. Of the 362 patients who had asymptomatic lower extremities, 128 (35%) had DVT. Moreover, clots were found in asymptomatic limbs in an additional 263 patients whose contralateral limb was symptomatic. Logistic regression analysis did not reveal combinations of symptoms and risk factors that could predict DVT. If DVT is suspected, the patient should undergo bilateral lower extremity duplex scanning.

Gallbladder volvulus in the elderly. An emergent surgical disease.

Volvulus of the gallbladder is an extremely rare condition presenting often in elderly patients commonly mimicking acute cholecystitis. Two cases of gallbladder volvulus in two octogenarian patients are presented from our institution. Clinical presentation is reviewed and some characteristic clinical and radiographic findings are described. The etiology of this rare entity is discussed with particular emphasis on visceroptosis, a common finding in elderly patients. The importance of early recognition and rapid treatment of this potentially fatal disease is emphasized. It is possible with increasing longevity, with its accompanying tendency to visceroptosis, that this entity may occur more frequently in the future. Its recognition and proper treatment is essential to good results in this elderly group of patients.

Wandering spleen.

The spleen is rather firmly attached in the left upper quadrant by five ligaments or peritoneal reflections. With congenital failure or acquired laxity of these attachments, the genital failure or acquired laxity of these attachments, the spleen acquires a true vascular pedicle and becomes mobile. This rare condition, called wandering spleen, makes the organ subject to the complication of torsion, which usually produces an acute abdominal emergency and requires immediate surgical removal. Symptomatic patients display a characteristic constellation of findings that strongly suggests the correct diagnosis and can definitively be ascertained by isotopic imaging specific for the spleen or by ultrasonography. Splenectomy should be performed for all cases of wandering spleen with significant symptoms. A conservative, nonoperative approach in asymptomatic patients is indicated to avoid any chance of postsplenectomy septicemia.