Desulfurization and denitrogenation of heavy gas oil by Rhodococcus erythropolis ATCC 4277.

Some of the noxious atmospheric pollutants such as nitrogen and sulfur dioxides come from the fossil fuel combustion. Biodesulfurization and biodenitrogenation are processes which remove those pollutants through the action of microorganisms. The ability of sulfur and nitrogen removal by the strain Rhodococcus erythropolis ATCC 4277 was tested in a biphasic system containing different heavy gas oil concentrations in a batch reactor. Heavy gas oil is an important fraction of petroleum, because after passing through, the vacuum distillation is incorporated into diesel oil. This strain was able to remove about 40% of the nitrogen and sulfur present in the gas heavy oil. Additionally, no growth inhibition occurred even when in the presence of pure heavy gas oil. Results present in this work are considered relevant for the development of biocatalytic processes for nitrogen and sulfur removal toward building feasible industrial applications.

Gymnasts utilize visual and auditory information for behavioural synchronization in trampolining.

In synchronized trampolining, two gymnasts perform the same routine at the same time. While trained gymnasts are thought to coordinate their own movements with the movements of another gymnast by detecting relevant movement information, the question arises how visual and auditory information contribute to the emergence of synchronicity between both gymnasts. Therefore the aim of this study was to examine the role of visual and auditory information in the emergence of coordinated behaviour in synchronized trampolining. Twenty female gymnasts were asked to synchronize their leaps with the leaps of a model gymnast, while visual and auditory information was manipulated. The results revealed that gymnasts needed more leaps to reach synchronicity when only either auditory (12.9 leaps) or visual information (10.8 leaps) was available, as compared to when both auditory and visual information was available (8.1 leaps). It is concluded that visual and auditory information play significant roles in synchronized trampolining, whilst visual information seems to be the dominant source for emerging behavioural synchronization, and auditory information supports this emergence.

Calcium depletion blocks the maturation of rotavirus by altering the oligomerization of virus-encoded proteins in the ER.

Maturation of rotavirus occurs in the ER. The virus transiently acquires an ER-derived membrane surrounding the virus particle before the eventual formation of double-shelled particles. The maturation process includes the retention and selective loss of specific viral protein(s) as well as the ER-derived membrane during formation of the outer capsid of the mature virus. When infected cells were depleted of Ca++ by use of the ionophore A23187 in calcium-free medium, membrane-enveloped intermediates were seen to accumulate. When Mn++, an efficient Ca++ competitor, was used to replace Ca++ in the medium, the accumulation of the enveloped intermediate was again observed, pointing to an absolute requirement of Ca++ in the maturation process. It was previously demonstrated in this laboratory that a hetero-oligomeric complex of NS28, VP7, and VP4 exists which may participate in the budding of the single-shelled particle into the ER (Maass, D. R., and P. H. Atkinson, 1990. J. Virol. 64:2632-2641). The present study demonstrates that either in the absence of Ca++ or in the presence of tunicamycin, a glycosylation inhibitor, VP7 is excluded from these hetero-oligomers. In the presence of Mn++, VP4 was blocked in forming a hetero-oligomeric complex with NS28 and VP7. The electrophoretic mobility of the viral glycoproteins synthesized in the presence of the ionophore were found to be altered. This size difference was attributed to altered N-linked glycosylation and carbohydrate processing of the viral glycoproteins. These results imply a major role for calcium and the state of glycosylation of NS28 in the assembly and acquisition of specific viral protein conformations necessary for the correct association of proteins during virus maturation in the ER.

An Evaluation of Kinetic Models in the Biodesulfurization of Synthetic Oil by Rhodococcus erythropolis ATCC 4277.

Biodesulfurization is an eco-friendly technology applied in the removal of sulfur from fossil fuels. This technology is based on the use of microorganisms as biocatalysts to convert the recalcitrant sulfur compounds into others easily treatable, as sulfides. Despite it has been studied during the last decades, there are some unsolved questions, as per example the kinetic model which appropriately describes the biodesulfurization globally. In this work, different kinetic models were tested to a batch desulfurization process using dibenzothiophene (DBT) as a model compound, n-dodecane as organic solvent, and Rhodococcus erythropolis ATCC 4277 as biocatalyst. The models were solved by ODE45 function in the MATLAB. Monod model was capable to describe the biodesulfurization process predicting all experimental data with a very good fitting. The coefficients of determination achieved to organic phase concentrations of 20, 80, and 100 % (v/v) were 0.988, 0.995, and 0.990, respectively. R. erythropolis ATCC 4277 presented a good affinity with the substrate (DBT) since the coefficients of saturation obtained to reaction medium containing 20, 80, and 100 % (v/v) were 0.034, 0.07, and 0.116, respectively. This kinetic evaluation provides an improvement in the development of biodesulfurization technology because it showed that a simple model is capable to describe the throughout process.

Burn trauma and tumor necrosis factor alpha alter calcium handling by cardiomyocytes.

It is well recognized that burn trauma induces an inflammatory cascade and the release of cytokines including tumor necrosis factor (TNF)-alpha. The negative inotropic effects of TNF-alpha on the heart are well recognized, but the cellular mechanisms remain unclear. To examine one aspect of cellular function, we exposed cardiac myocytes isolated from NZW rabbits (collagenase digestion) to either TNF-alpha (200, 400, or 1000 U/mL) or sham or burn plasma (10% by volume) for 3 to 4 h and measured calcium transient ratios in the isolated, contracting myocytes using the fluorescent indicator Fura-2-acetoxymethyl (1.2 microM); myocytes treated with media alone served as controls. Cells were placed in a perfusion chamber on the stage of an inverted Nikon microscope and superfused with buffer at 37 degrees C and stimulated at 1 Hz. A Tracor Northern Fluoroplex 1000 microspectrofluorometer and camera system, set to provide excitation of 340 and 380 nm with emission at 450-580 nm, was used to measure Ca2+ transients during systole-diastole. [Ca2+]i was reported as a fluorescence ratio (F340/F380) to minimize effects of different cell thickness and motion artifacts. After recording diastolic/systolic [Ca2+]i, cells were stimulated with isoproterenol, and [Ca2+]i was again measured. TNF-alpha produced diastolic and systolic [Ca2+]i values (1.067 +/- .023/1.301 +/- .017) that were similar to values seen after myocyte exposure to burn plasma (1.099 +/- .024/1.307 +/- .028) and significantly greater than values measured in controls (.857 +/- .017/1.077 +/- .015, p < .05). Our data confirm that burn trauma and TNF-alpha alter calcium handling by cardiomyocytes. The possible contribution of altered intracellular calcium dynamics to cardiac contractile abnormalities after burn trauma and TNF-alpha administration warrants further study.

Development of an acute burn model in adult mice for studies of cardiac function and cardiomyocyte cellular function.

The increasing availability of mice with gene supplementation (transgenic), site-specific inactivation mutations (gene "knock-outs"), or site-specific genetic modification mutations (gene "knock-ins") has spurred interest in the development of murine trauma models. In this study, C57 BL/6 mice (28 g) were given a cutaneous burn over 40% total body surface area by applying brass probes (1 x 2 x 0.003 cm) heated to 100 degrees C in boiling water to the animals side and back for 5 s. Shams received anesthesia alone and not burn. Mice were killed 24 h post-burn to determine presence of partial-thickness or full-thickness burn injury, cardiac contractile function (Langendorff perfusion, n = 7 or 8 mice/group) or to examine cardiac myocyte cytokine secretion in isolated cardiomyocytes (collagenase perfusion, n = 4 or 5 mice/group). All mice were killed 24 h post-burn for subsequent cardiac or cardiomyocyte studies. Our studies confirm that this murine model of burn trauma produced mixed partial- or full-thickness burn injury, whereas there was no necrosis or inflammation in sham burn mice. Baseline hematocrits were similar in all mice (44+/-1) but decreased after burn trauma (37+/-1), likely because of the volume of fluid resuscitation and hemodilution. Burn trauma impaired cardiac contraction and relaxation as indicated by the lower left ventricular pressure (LVP) measured in burn (56+/-4) compared to that measured in shams (84+/-1 mmHg, P < 0.001), a lower rate of LVP rise (+dP/dt max, 1393+/-10 vs. 2000+/-41 mmHg/s, P < 0.002), and reduced LVP fall (-dP/dt max, 1023 - 40 vs. 1550+/-50, P < 0.001). These differences occurred despite similar coronary perfusion pressures and heart rates in both sham and burn mice. Ventricular function curves were shifted downward in the burn mice in the direction of contractile failure; in addition, hearts from burn mice had reduced LVP and +dP/dt responses to increases in coronary flow rate, increases in perfusate Ca2+, and to isoproterenol challenge (P < 0.05). Burn trauma promoted cardiac myocyte secretion of tumor necrosis factor (TNFalpha) (175+/-6 pg/mL) compared to that measured in shams (72+/-9 pg/mL, P < 0.05); burn trauma also increased cardiac myocyte secretion of interleukin 1beta (IL-1beta) (sham: 2+/-0.5; burn: 22+/-1 pg/mL, P < 0.05) and IL-6 (sham: 70+/-6; burn: 148+/-16 pg/mL, P < 0.05). Anti-TNFalpha strategies prevented burn-mediated cardiac contractile deficits. Burn trauma altered Ca2+ homeostasis in murine cardiomyocytes (Fura-2 AM loading). [Ca2+]i in myocytes from burns (185+/-4 nM) was higher than values measured in myocytes from shams (86+/-nM, P < 0.05). These data confirm that the murine burn model provides a reasonable approach to study the molecular and cell biology of inflammation in organ dysfunction after burn trauma.

Major burn trauma in rats promotes cardiac and gastrointestinal apoptosis.

The hypothesis that cardiac functional abnormalities that occur after major burn trauma are paralleled by an increased incidence of apoptosis in cardiac myocytes was examined. Adult Sprague-Dawley rats were given a full thickness scald burn comprising 43+/-1% of the total body surface area or were manipulated identically but not exposed to burn injury (sham burn); burned rats were fluid resuscitated with lactated Ringer's solution. Tissues from burn and sham burn animals were then examined by the TUNEL (TdT-mediated dUTP nick end labeling) assay and light microscopy to determine the presence of apoptosis 24 and 48 h after burn trauma. In parallel, the mechanical function of the heart was assayed in separate groups of rats. Tissues harvested from the hearts of sham-treated animals showed essentially no apoptosis, whereas a small number of apoptotic cells were noted in the intestinal villi and liver of sham-treated animals. Twenty-four hours after burn trauma, there was a marked increase in apoptotic cells in the left ventricle (+916%), and the number of apoptotic cells remained increased by eightfold 48 h postburn. Apoptosis was noted predominately in the subendocardial tissue of the left ventricle. The appearance of apoptotic cells was paralleled by a decrease in cardiac mechanical function with significant decreases in left ventricular pressure and +/-dP/dt(max). Burn injury also increased apoptosis in the small intestine significantly, whereas apoptosis in the liver did not increase with burn trauma. These data suggest that the apoptosis of the cardiac myocytes that occurs after burn trauma may contribute, in part, to postburn cardiac mechanical dysfunction.

Time course of myocardial sodium accumulation after burn trauma: a (31)P- and (23)Na-NMR study.

In this study, (23)Na- and (31)P- nuclear magnetic resonance (NMR) spectra were examined in perfused rat hearts harvested 1, 2, 4, and 24 h after 40% total body surface area burn trauma and lactated Ringer resuscitation, 4 ml. kg(-1). %(-1) burn. (23)Na-NMR spectroscopy monitored myocardial intracellular Na+ using the paramagnetic shift reagent thulium 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylenephosphonic acid). Left ventricular function, cardiac high-energy phosphates (ATP/PCr), and myocyte intracellular pH were studied by using (31)P NMR spectroscopy to examine the hypothesis that burn-mediated acidification of cardiomyocytes contributes to subsequent Na+ accumulation by this cell population. Intracellular Na+ accumulation was confirmed by sodium-binding benzofuran isophthalate loading and fluorescence spectroscopy in cardiomyocytes isolated 1, 2, 4, 8, 12, 18, and 24 h postburn. This myocyte Na+ accumulation as early as 2 h postburn occurred despite no changes in cardiac ATP/PCr and intracellular pH. Left ventricular function progressively decreased after burn trauma. Cardiomyocyte Na+ accumulation paralleled cardiac contractile dysfunction, suggesting that myocardial Na+ overload contributes, in part, to the progressive postburn decrease in ventricular performance.

Arginine in burn injury improves cardiac performance and prevents bacterial translocation.

This study examined the effects of arginine supplement of fluid resuscitation from burn injury on cardiac contractile performance and bacterial translocation after a third-degree burn comprising 43% of the total body surface area in adult rats. Before burn injury, rats were instrumented to measure blood pressure; after burn (or sham injury), paired groups of sham-burned and burned rats were given vehicle (saline), L-arginine, D-arginine, or N-methyl-L-arginine (300 mg/kg in 0.3 ml of saline 30 min, 6 h, and 23 h postburn) plus fluid resuscitation; sham-burned rats received drug only. Twenty-four hours after burn trauma, hemodynamics were measured; the animals were then killed and randomly assigned to Langendorff heart studies or to studies examining translocation of gut bacteria. Burn rats treated with vehicle, D-arginine, or N-methyl-L-arginine had well-defined cardiocirculatory responses that included hypotension, tachycardia, respiratory compensation for metabolic acidosis, hypocalcemia, cardiac contractile depression, and significant bacterial translocation. Compared with values measured in vehicle-treated burn rats, L-arginine given after burn improved blood pressure, prevented tachycardia, reduced serum lactate levels, improved cardiac performance, and significantly reduced bacteria translocation, confirming that L-arginine administration after burn injury provided significant cardiac and gastrointestinal protection. Circulating neutrophil counts fell after burn trauma and serum glucagon levels rose, but these changes were not altered by pharmacological intervention. Our finding of significantly higher coronary perfusate guanosine 3',5'-cyclic monophosphate concentration in L-arginine-treated burn rats suggests that the beneficial effects of L-arginine were mediated by nitric oxide production.

End stage coronary disease treated with the transmyocardial CO2 laser revascularization: a chance for the 'inoperable' patient.

OBJECTIVE: The aim of this study is to evaluate the short and mid-term efficacy of the Transmyocardial High Power CO2 Laser Revascularisation (TMLR) as a last resource method for end-stage coronary disease patients. METHOD AND PATIENTS: The High Power CO2 Laser 800 W Heart Laser (PLC Medical Systems) was used since February 1994 to treat 268 patients. In 52% of the cases (140) the indication for TMLR treatment was virtual inoperability by the classical bypass revascularisation. In the other 128 patients (48%), where only an incomplete revascularisation was expected, the TMLR was combined with a feasible bypass revascularisation (CABG). Of all patients, 71% were operated on 1-5 times before and or treated by several percutaneous transluminal coronary angioplasty (PTCA). All patients were sufferers of angina pectoris and most were classified Canadian Cardiac Society (CCS) 3-4, despite the maximal medical treatment. The ejection fraction was normal in 13% of patients only, and in 47% of them it was below 40% (10-68%). RESULTS: The operation itself was generally well tolerated. We lost only one patient at the table. The hospital survival was 89.2%; 88.2% in the combined group and 90.3% in the TMLR only group. After the routine follow up screening 3, 6 and 12 months postoperatively (262 patients--131 TMLR and 131 TMRL/CABG), 40% of the TMLR patients upgraded into the functional class CCS 0-1; the combined group of patients scored up even in 84%. All considering their quality of life to be 'better than years ago'. The ergometry stress test, impossible for most of them before, became feasible and better in 80% of the patients. In the follow up period of the combined group, another 6 (4.7%), and in the TMLR only group, 12 (9.4%) patients died. CONCLUSION: The short and middle term results of this--until now the largest single institution series of TMLR treated patients--were that patients almost without exception were refused for any kind of surgery by several other centres; this shows an acceptable survival rate and a surprising level of pain relief, increased activity and better quality of life then ever expected. In our experience, TMLR is a suitable method for treatment of end stage coronary disease, if all standard measures, medical therapy, PTCA and redo coronary revascularisation possibilities are exhausted. The favourable results imply the question as to whether this method will become an alternative for a second bypass operation in the future. The TMLR as an alternative for heart transplant is already a reality for some of our patients.

Digital 3D image reconstruction of ventriculocapillary communication as revealed in one case after transmyocardial laser revascularization.

TMR (Transmyocardial Laserrevascularization) was performed on the partially dyskinetic left ventricular anterior wall with stenotic coronary blood supply in a 61 year old woman with a history of angina and myocardial infarction. As an ischemic aneurysm developed in the anteroapical region of the TMR treated area, it became clear that TMR did not provide a substitute for coronary blood supply in this very heart region. The aneurysm was removed surgically 7 months after TMR and showed histopathologic features of an acute aneurysm. Three-dimensional image analysis helped prove the presence of linear tracks through several serial sections which were not easily visible in routine histology sections. Also, three-dimensional vessel reconstruction showed a connection between a small endocardial pit on one serial section with the capillary network in the adjacent serial sections. The results should not be generalized, as currently aneurysmectomy is an end point not reached by the majority of TMR-treated patients.

Successful replantation of a hand amputated through the metacarpus.

A successful replantation is reported of a hand completely severed by a circular saw through all five metacarpals. The sequence of primary reconstruction of all important structures beginning three hours after the injury and the functional results eighteen months later are presented.

The helix filter: a new vena caval filter for the prevention of pulmonary embolism.

The helix caval filter is a continuous band spiral composed of a highly elastic metal alloy. A vertical double-helix coil fixes the filter in the infrarenal vena cava by elastic expansion while a watch spring type horizontal coil works as the filtering element. The diameter of the filter can be reduced by torsion from 32 to 7 mm. Transvenous implantation was performed under fluoroscopy using a newly developed instrument featuring intraoperative angiographic control, high flexibility, a guidance system, and a well-controlled release mechanism. The filter and the implantation technique were tested in a circulation system and in 10 dogs and 4 calves (follow-up 3-91 days). In addition, two filters were released intentionally into the right atrium of a dog and a calf. Flow resistance of the filter is negligible up to more than 10 1/min. Thrombi larger than 3.5 mm were filtered out reliably. Lethal complications, filter migrations or perforations did not occur in animal experiments. Filter coils which are in contact with the vessel wall become firmly incorporated within 3 to 4 weeks. The patency rate of the vena cava was 80% (8/10) for dogs and 100% (4/4) for calves. Implantations in the right atrium did not cause any symptoms. The filter's design allows fixation without hooks or spikes and consequently makes transvenous extraction possible up to 7-10 days after implantation. Comparable clinical results could extend the indications for caval filter implantation, and the decision which is still difficult today could be made easier.

Treatment of major venous obstruction with an expandable endoluminal spiral prosthesis.

Six patients with extensive iliofemoral and/or caval thrombosis were treated by thrombectomy and subsequent implantation of an expandable endoluminal spiral prosthesis in case of congenital caval stenoses (1) or extravascular compression or traction (5). Five of these patients had undergone previous surgery and thrombosis had developed despite low dose heparin given postoperatively. Three patients had had an unsuccessful thrombectomy prior to spiral implantation. No complications related to the endoluminal prosthesis occurred. There was one retroperitoneal hematoma from guide wire perforation of the inferior vena cava (IVC) necessitating laparotomy and reversal of the previously constructed femoral av-fistula with subsequent iliofemoral rethrombosis. All other endoluminally reconstructed veins remained open at early and late (up to 12 months) review confirmed by phlebography. We conclude that with increasing clinical experience endoluminal relining of obstructed major veins will probably become a valuable method of venous reconstruction with minimal surgical trauma.

Initial clinical experience with an endoluminal spiral prosthesis for treating complicated venous thrombosis and preventing pulmonary embolism.

Fourteen patients with complicated venous thrombosis or recurrent pulmonary embolism were treated by implantation of an endoluminal spiral prosthesis subsequent to balloon angioplasty, surgical thrombectomy or embolectomy, a combination of these, or, in 2 cases, no other treatment. The patients were divided into 2 groups, based on their primary diagnosis and the purpose of the prosthesis. Group I included 8 patients with extensive iliofemoral or caval thrombosis, caused by congenital caval stenosis (1 case) or extravascular compression or retraction (7 cases); 7 of these patients had had previous operations, and the remaining patient had undergone thrombolysis, which failed. The current treatment consisted of balloon angioplasty and surgical thrombectomy or embolectomy, and implantation of an endoluminal spiral stent to prevent elastic recoil of the vessel. In 4 cases, an arteriovenous fistula was constructed and was taken down 3 months later; in 1 additional patient, a bilateral arteriovenous fistula was created. Group II comprised 6 patients with recurrent pulmonary embolism (4 cases), massive pulmonary embolism (1 case), or paradoxical bilateral carotid artery embolism (1 case). Four of these patients underwent surgical thrombectomy or embolectomy, while 2 had no treatment other than filter implantation. All 6 underwent transluminal implantation of a helix caval filter (a modification of the endoluminal spiral stent). All but 1 implantation was accomplished by means of either a transfemoral or a transjugular cutdown; the remaining implantation was performed transatrially after a pulmonary embolectomy. The only device-related complication was a retroperitoneal hematoma in Group I, resulting from perforation of the inferior vena cava by the guidewire during device implantation. This complication necessitated an emergency laparotomy and takedown of the arteriovenous fistula, which resulted in rethrombosis of the left iliofemoral vein. The other 7 stented veins were patent at early phlebographic follow-up, as were all 5 of those studied later. One Group-I patient died 4 months after surgery, due to tumor progression and without signs of caval restenosis. Twelve months postoperatively, 1 Group-II patient died of urosepsis without a recurrence of pulmonary embolism. Four of the 6 Group-II patients were studied late postoperatively, and all of their stented vessels were patent. There was no operative mortality or postoperative embolism. On the basis of these results, we conclude that endoluminal stenting with an expandable spiral prosthesis is a promising method for remote venous reconstruction. Moreover, it appears that the modified stent, or helix caval filter, compares favorably with commercially available filters.

[Groin flap plastic surgery using McGregor's method]. / Die Leistenlappenplastik nach McGregor.

The specific advantages of the groin flap to the other axial pattern flaps from the trunk are outlined, followed by a detailed description of the operative procedure and the postoperative management. Based on our experience, the groin flap is an uncritical flap and can be recommended in numerous instances either of primary repair or secondary reconstruction of the injured hand.

Murein biosynthesis in ether permeabilized Escherichia coli starting from early peptidoglycan precursors.

ETB, ether treated bacteria, from E. coli and other Gram-negative strains, contain in a cell-free system all enzymes necessary for murein biosynthesis. Starting with a variety of combinations of peptidoglycan precursors, high yields of sodium dodecylsulfate (SDS, 4%) insoluble murein or murein like material were synthesized. The amount of newly synthesized SDS insoluble material (NSM) was dependent upon the growing phase at which cells had been harvested for preparation of ETB. This data may provide some insight into the regulation of peptidoglycan biosynthesis. Starting from early peptidoglycan precursors, the cell-free synthesis of NSM was inhibited by specific inhibitors of murein synthesis, such as D-cycloserine, D-fluoroalanine, 2-amino-ethylphosphonate, analogues of D-alanyl-D-alanine and beta-lactam antibiotics at appropriate concentrations. Some D-alanyl-D-alanine analogues and 4-chlorodiaminopimelic acid were incorporated into NSM in place of their corresponding natural substrates.

Retention by the endoplasmic reticulum of rotavirus VP7 is controlled by three adjacent amino-terminal residues.

The rotavirus outer capsid glycoprotein, VP7, is an endoplasmic reticulum (ER) membrane-associated glycoprotein in both infected and transfected cells. It was previously demonstrated in this laboratory and by others that both the cleaved signal sequence (H2) and the first NH2-terminal 61 amino acids of VP7 are sufficient and necessary for ER retention of this molecule. Using site-specific mutagenesis and transfection techniques, we show that residues Ile-9, Thr-10, and Gly-11 were specifically necessary for ER retention. These results further define the ER retention sequence of VP7 and demonstrate that conservative changes, apparently innocuous in only three adjacent amino acids, can lead to major solubility and compartmentalization changes. It was found that placement of the first 31 mature NH2-terminal residues of VP7, in addition to the cleaved ER translocation signal sequence, was sufficient to retain the enzymatically active chimeric alpha-amylase in the ER; this enzyme is normally secreted. Deletions of the residues Ile-9, Thr-10, and Gly-11 within the amylase chimera containing 31 VP7 amino acids resulted in secretion of enzymatically active protein. It was also observed that the residues of VP7 presented in certain chimeras were able to abolish alpha-amylase enzymatic activity. These chimeras are presumably misfolded since it was demonstrated by pulse-chase experiments that these molecules are degraded in the ER. We surmise that a favorable conformation is necessary for retention since ER retention and activity of the chimeras depend on the primary sequence context.