Exploration of surfactin production by newly isolated Bacillus and Lysinibacillus strains from food-related sources.

As a lipopeptide (LP), surfactin exhibits properties, such as emulsifying and dispersing ability, which are useful in food industry. Discovery of new LP-producing strains from food sources is an important step towards possible application of surfactin in foods. A total of 211 spore-forming, Gram-positive, and catalase-positive bacterial strains were isolated from fermented African locust beans (iru) and palm oil mill effluents in a screening process and examined for their ability to produce surfactin. This was achieved by a combination of methods, which included microbiological and molecular classification of strains, along with chemical analysis of surfactin production. Altogether, 29 isolates, positive for oil spreading and emulsification assays, were further identified with 16S rDNA analysis. The strains belonged to nine species including less commonly reported strains of Lysinibacillus, Bacillus flexus, B. tequilensis, and B. aryabhattai. The surfactin production was quantitatively and qualitatively analysed by high-performance thin-layer chromatography and liquid chromatography-mass spectrometry (LC-MS). Confirmation of surfactin by MS was achieved in all the 29 strains. Highest surfactin production capability was found in B. subtilis IRB2-A1 with a titre of 1444·1 mg L-1 .

Geology of the InSight landing site on Mars.

The Interior Exploration using Seismic Investigations, Geodesy and Heat Transport (InSight) spacecraft landed successfully on Mars and imaged the surface to characterize the surficial geology. Here we report on the geology and subsurface structure of the landing site to aid in situ geophysical investigations. InSight landed in a degraded impact crater in Elysium Planitia on a smooth sandy, granule- and pebble-rich surface with few rocks. Superposed impact craters are common and eolian bedforms are sparse. During landing, pulsed retrorockets modified the surface to reveal a near surface stratigraphy of surficial dust, over thin unconsolidated sand, underlain by a variable thickness duricrust, with poorly sorted, unconsolidated sand with rocks beneath. Impact, eolian, and mass wasting processes have dominantly modified the surface. Surface observations are consistent with expectations made from remote sensing data prior to landing indicating a surface composed of an impact-fragmented regolith overlying basaltic lava flows.

Kinetic analysis and modeling of the liquid-liquid conversion of emulsified di-rhamnolipids by Naringinase from Penicillium decumbens.

The enzymatic conversion of an aggregate-forming substrate was kinetically analyzed and a model was applied for the prediction of reaction-time courses. An L-rhamnose molecule from a di-rhamnolipid is cleaved by Naringinase from Penicillium decumbens leading to a mono-rhamnolipid. Optimal reaction rates were found when both, substrate and product build large co-aggregates in a slightly acidic aqueous phase. On the other hand, reaction rates were independent of initial di-rhamnolipid concentration and this was interpreted by assuming that the reaction occurs in the aqueous phase according to Michaelis-Menten kinetics in combination with competitive L-rhamnose inhibition. Rhamnolipids were therefore assumed to be highly concentrated in aggregates, a second liquid phase, whereas diffusive rhamnolipid transport from and to the aqueous phase occurs due to the enzymatic reaction. Furthermore, ideal surfactant mixing between di- and mono-rhamnolipid was assumed for interpretation of the negative effect of the last on the reaction rate. A model was created that describes the system accordingly. The comparison of the experimental data, were in excellent agreement with the predicted values. The findings of this study may beneficially be adapted for any bioconversion involving aggregate-forming substrate and/or product being catalyzed by hydrophilic enzymes.

Non-porous magnetic micro-particles: comparison to porous enzyme carriers for a diffusion rate-controlled enzymatic conversion.

In this study the kinetics of conversion of a low-soluble substrate by an immobilized enzyme was investigated with respect to the diffusion limitation within porous and non-porous carriers. Non-porous micro-magnetic beads in comparison to conventional porous supports like Eupergit and Sepharose were tested. Due to their small diameters and their magnetic properties, micro-magnetic beads are especially applicable in diffusion rate-controlled processes in biological suspensions. The enzymatic reaction studied was the conversion of emulsified dirhamnolipid by immobilized Naringinase from Penicillium decumbens to monorhamnolipid and L-rhamnose. Taking into account mass transfer phenomena, the variation of the reaction effectiveness factor with increasing enzyme loading was estimated and compared with experimental efficiencies utilizing different enzyme loaded immobilized preparations. For comparison, carrier activities were also determined with the model substrate p-nitro-phenyl-rhamnoside. Intrinsic enzyme activities were thereby evaluated for porous supports. Highest specific activities were obtained with the micro-magnetic beads. These non-porous micro-beads demonstrated to be the most suitable carrier for bioconversion of a low-soluble substrate like rhamnolipids, where mass diffusional resistances in the three-phase reaction process are completely overcome. However, the smaller particle surface available limited the specific activity obtained at high protein loadings.

Biosurfactant synthesis by Pseudomonas aeruginosa LBI isolated from a hydrocarbon-contaminated site.

AIMS: Pseudomonas aeruginosa LBI (Industrial Biotechnology Laboratory) was isolated from hydrocarbon-contaminated soil as a potential producer of biosurfactant and evaluated for hydrocarbon biodegradation. The emulsifying power and stability of the product was assessed in the laboratory, simulating water contamination with benzene, toluene, kerosene, diesel oil and crude oil at various concentrations. METHODS AND RESULTS: Bacteria were grown at 30 degrees C and shaken at 200 rpm for 168 h, with three repetitions. Surface tension, pH and biosurfactant stability were observed in the cell-free broth after 168 h of incubation. The strain was able to produce biosurfactant and grow in all the carbon sources under study, except benzene and toluene. When cultivated in 30% (w/v) diesel oil, the strain produced the highest quantities (9.9 g l(-1)) of biosurfactant. The biosurfactant was capable of emulsifying all the hydrocarbons tested. CONCLUSION: The results from the present study demonstrate that Ps. aeruginosa LBI can grow in diesel oil, kerosene, crude oil and oil sludge and the biosurfactant produced has potential applications in the bioremediation of hydrocarbon-contaminated sites. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas aeruginosa LBI or the biosurfactant it produces can be used in the bioremediation of environmental pollution induced by industrial discharge or accidental hydrocarbon spills.

Methylation of DNA.

The methylated bases of DNA are formed by the transfer of the methyl group from S-adenosylmethionine to a polynucleotide acceptor. This transfer is catalyzed by highly specific enzymes which recognize a limited number of available sites in the DNA. The mechanism for the recognition is presently unknown. In some instances, there is evidence that other cellular components, such as lipopolysaccharides, can influence the methylation reaction. Certain bacteriophages induce new methylases upon infection of their hosts. Phage T3 is unique in establishing an environment in which methylation of neither the phage nor the host nucleic acid can occur. By superinfecting T3-infected cells with other phages, the latter can be obtained with methyl-deficient DNA. Although a great deal is known about the enzymology of the methylation reaction, and there appears to be a strong correlation between the in vitro and in vivo reactions, studies in which DNA is either supermethylated or totally unmethylated have not yielded any insight as to what the possible function of the methylated bases may be.

Rhamnolipids know-how: Looking for strategies for its industrial dissemination.

Despite the numerous advantages of biosurfactants, such as low toxicity, biodegradability and high stability, these compounds are not widely used because of the high cost of production. Details about genetics, regulation and biosynthesis of rhamnolipids by Pseudomonas aeruginosa, are extremely important to the development of bioprocesses involving the synthesis of these compounds. The holding of such knowledge associated with the use of metabolic engineering tools allow modification of producing strains and the development of synthetic routes, with the purpose of increasing the production of rhamnolipids. Considering the need to obtain this know-how, this review provides information on the rhamnolipids, covering genetics, biosynthesis of hydrophobic and hydrophilic portions, and regulation, plus some future strategies that would contribute to the expansion of the production of this green surfactant.

Adjuvant intravesical mitoxantrone after transurethral resection of primary superficial transitional cell carcinoma of the bladder. A prospective randomised study.

A prospective randomised controlled clinical trial began in 1989 on 126 patients with superficial transitional cell carcinoma of the bladder (pTa-pT1, grades 1-3) to compare the efficacy of adjuvant topical mitoxantrone after transurethral resection versus no further treatment. 62 patients received no further treatment, 64 patients received weekly 20 mg mitoxantrone intravesically for 6 weeks after differentiated TUR of all visible tumours. The endpoint of the study was any progression of stage or grade or further recurrences. The median follow up was 29 months--the minimum follow up was 24 months. The percentage of recurrences (25.8 versus 23.4), the recurrence rate (1.2 versus 0.9), the overall disease free interval and the tumour progression rate showed no statistically significant differences (P > 0.05 Mantel-Cox test). Only the comparison of time to recurrence in tumours with recurrences showed a statistically significant difference, with a longer disease free interval for the TUR plus mitoxantrone group (P = 0.016 Mantel-Cox test).

Inhibition of anti-HLA-B7 alloreactive CTL by affinity-purified soluble HLA.

The objective of this study was to elucidate the interaction of naturally occurring soluble MHC class I molecules with alloreactive CTL and to discuss its possible relevance to graft acceptance. An anti-HLA-B7 specific CTL-line, BV.B7, was generated in vitro. On phenotyping the cells after 6 weeks, 80% were found to be CD8+, 14% CD4+ and 6% CD8+CD4+. CD4+ CTL were depleted using immunomagnetic beads precoated with an anti-CD4 antibody. Of the recovered CTL greater than 96% were CD8+. A total of 12 HLA-B7 target cell lines and PHA blasts tested were specifically lysed in a 51Cr-release assay. Soluble HLA class I molecules were isolated on affinity chromatography columns using the anti-HLA-B7 ME 1 and the anti-heavy chain W6/32 monoclonal antibodies. Antigen purity was confirmed by analysis on SDS-PAGE gels. CTL were preincubated with 0.1-1.8 micrograms/ml soluble HLA for 30 min at 37 degrees C and subsequently tested for cytotoxicity in the 51Cr-release assay; 1.1 micrograms/ml HLA-B7 molecules reduced CTL cytotoxicity by 50% whereas non-B7 HLA had no effect. Further, CTL cytotoxicity was reduced by preincubation with anti-CD8, anti-TcR, and anti-CD3 antibodies. We anticipate a possible down-regulatory role of soluble HLA on CTL in allogeneic transplantation.

Diagnosis and staging of head and neck cancer: a comparison of modern imaging modalities (positron emission tomography, computed tomography, color-coded duplex sonography) with panendoscopic and histopathologic findings.

OBJECTIVE: To compare the clinical value of positron emission tomography (PET) using fludeoxyglucose F 18, computed tomography (CT), color-coded duplex sonography (CCDS), and panendoscopy in the detection and staging of head and neck cancer. DESIGN: Prospective nonrandomized controlled study. SETTING: Medical school. PATIENTS: Convenience sample of 50 patients with suspected primary or recurrent head and neck cancer. INTERVENTION: Biopsy, tumor surgery. MAIN OUTCOME MEASURES: Information of diagnostic procedures compared with histopathologic features. RESULTS: Both PET and panendoscopy had a sensitivity of 95% and 100% for detection of primary tumor or recurrent carcinomas, respectively. Specificity for PET and panendoscopy was 92% and 85% in primary tumors and 100% and 80% in recurrent carcinoma, respectively. Sensitivity of CCDS and CT was 74% and 68% in primary tumors and 67% and 63% in recurrent carcinomas, respectively. Specificity was 75% and 69% in primary tumors and 100% and 80% in recurrent neoplasms. When assessing neck nodes, all imaging procedures exhibited identical sensitivity (84%). Specificity was 90%, 96%, and 88% in PET, CT, and CCDS, respectively. In recurrent lymph node metastases, sensitivity was 100%, 67%, and 67% and specificity was 87%, 91%, and 87% for PET, CT, and CCDS, respectively. CONCLUSIONS: Positron emission tomography was the most reliable imaging procedure in the detection of primary tumor and recurrent carcinomas localized in the head and neck region. Owing to its limited anatomical depiction, it cannot as yet replace other diagnostic procedures in preoperative planning but does contribute valuable complementary diagnostic information. Computed tomograpy may have difficulties in identifying recurrent carcinomas. For routine diagnosis of nodal spread in the neck, CCDS is recommended. Panendoscopy is a valuable diagnostic procedure that can provide key information in cases of superficial mucosal tumor involvement. Arch Otolaryngol Head Neck Surg. 2000;126:1457-1461

[The evaluation of 2D- and 3D-"time of flight" magnetic resonance angiography (MRA) in the diagnosis of renal artery stenoses]. / Bewertung der 2D- und 3D-"Time-of-flight"-Magnetresonanz-Angiographie (MRA) in der Diagnostik von Nierenarterienstenosen.

This study was carried out to evaluate time-of-flight RA in renal artery stenosis (RAS) in selected hypertensive patients (n = 41). In i.a. DSA studies, 10 unilateral, 8 bilateral RAS, and 4 unilateral RA occlusions were proven. MRA was done in coronal and axial 2D technique (FLASH), and in 3D technique (FISP) using GE-pulse sequences. DSA results were correlated with both 2D-individual slices, 2D- and 3D-MIP angiograms. Highest sensitivity and specificity was found for the axial 2D individual slice analysis (88%, 85% resp.), followed by the 3D-MIP MRA (78%, 80% resp.), and axial 2D-MIP MRA (73%, 79% resp.). MRA of renal arteries used in this study shows to be not adequate to DSA results due to many drawbacks. All MRA techniques, in particular the 3D-technique, tend to overestimate RAS occasionally pretending occlusions.

[The use of the RACE technique for quantitative flow measurements. Evaluation using a clinically relevant flow model]. / Einsatz der RACE-Technik zur quantitativen Flussmessung. Evaluierung an einem klinisch relevanten Flussmodell.

A phantom has been designed for measuring flow velocities; using pipes of specified diameter and selectable pulse rates, flow rates varying from 0-250 cm/s can be determined. The measurements are made in a superconductive magnet with a field strength of 1.5 Tesla, employing a RACE technique. The reference technique was electromagnetic flow measurement. There was good linear correlation between the electromagnetic and the MRT results with a correlation coefficient of 0.98. It is necessary in the first place to estimate flow speed; if flow velocity is unknown initially, there is no correlation. Hence, in everyday use erroneous results may be obtained, particularly since one cannot always demonstrate vessels in all parts of the body without superimposition. Further clinical studies are required in various vascular territories to evaluate the usefulness of the RACE technique for flow measurements.

[MR angiography. Its use in pulmonary and mediastinal space-occupying lesions]. / MR-Angiographie. Einsatz bei pulmonalen und mediastinalen Raumforderungen.

MR angiography (MRA) proved to be promising combined to MR imaging (MRI) in the assessment of intrathoracic masses. Sequential FLASH 2D angiograms were acquired in breath-hold technique using the following parameters: TR = 30 ms, TE = 10 ms, FA = 30 degrees. Section thickness was 5 mm with 1 mm overlap between sequential sections. Individual conditions of the examination were achieved by an automatized control procedure. Targeted MIP postprocessing resulted in 3D reconstructions illustrating vascular anatomy and avoiding superimposition. Presentation should be done by cine-mode for better spatial impression. This method was evaluated in a prospective study of 21 patients with malignant pulmonary and mediastinal masses in addition to spin-echo imaging. The diagnostic contribution concerning the relationship between the mass and the vasculature like displacement, stenosis, and poststenotic perfusion defect were assessed.

Thermally induced entrance wound-like defect of the skull.

A case of death due to car fire is described where the carbonised skull of the cremated corpse showed an ovoid defect of the cranial bone. The question arose whether this formed fracture was caused by a gunshot. But the reconstruction of the cranial remains with detached parts of the tabula externa provided evidence that the suspicious defect was to be classified as an uncommon heat-induced post-mortem artefact.

Suicide by a power drill.

This is the report of a 62-year-old man who committed suicide by drilling through his anterior chest wall with an electric power drill. Death was caused by pericardial tamponade combined with bleeding into the pleural cavity. The skin lesion at the left hemithorax was similar to a bullet entrance wound.

A contribution to a possible differentiation between SIDS and asphyxiation.

The singular and combined appearance of conjunctival petechiae, of acute pulmonary emphysema and of signs of aspiration was evaluated in resuscitated and non-resuscitated children dying of SIDS (n = 115), of other causes of natural death (n = 17), of severe head injury (n = 10) and of asphyxiation/strangulation (n = 7). Conjunctival petechiae occurred in all of the asphyxiated victims and in five out of 10 children dying of severe head injury, but were exclusively found in four resuscitated babies and one baby with questionable resuscitation trials of the SIDS-group (4%), furthermore in one resuscitated child dying of a congenital heart defect. Acute pulmonary emphysema was detected in six out of the seven children of the asphyxiation/strangulation group, in two resuscitated children with head injury and in five resuscitated babies dying of SIDS. Both parameters were observed exclusively in the asphyxiation/strangulation group (71% of the cases) but not in the control cases. Therefore, it can be concluded that the simultaneous appearance of conjunctival petechiae and of acute pulmonary emphysema strongly indicates death by asphyxiation.

Fractures at the base of the skull in gunshots to the head.

The distribution of fractures at the base of the skull was investigated in 147 victims with lethal head shot wounds caused by handguns or small calibre low-velocity rifles. In individuals without an impact of the projectile at the base of the skull, bullets lodged in the head were found up to a calibre of 7.65 mm (pistol) or .38 special (revolver), respectively. In cases with a trajectory through the cranial fossae positive results were obtained up to 9 mm (pistol) or even .45 (revolver). Fractures in anterior parts of the base of the skull were a rather frequent finding (82% of the cases) and could also be observed in victims shot by low energy guns. Fracture lines in particular in all cranial fossae, however, indicate a comparatively high energy missile and were found in victims without a direct impact of the bullet at the base of the skull after the use of guns with a calibre of at least 7.65 mm. Severe fractures leading to a hinge-like mobility of the base of the skull point also to projectiles of rather high energy. On the other hand, such fractures were also found in a victim with a contact shot by a .22 rifle. Therefore, it must be emphasized that individual factors such as the constitution of the base of the skull, the path of the bullet, technical parameters of the gun and ammunition used are of great importance if conclusions are to be drawn on an unknown gun by evaluation of fractures at the base of the skull.

Genetic polymorphism of alpha-2-HS-glycoprotein in northern Bavaria (Germany). Simplified AHSG-phenotyping by isoelectric focusing using dry gels.

A simple and practical method of detecting AHSG using isoelectric focusing on dry gels is described. The procedure is both rapid and provides reliable results. AHSG phenotypes were determinated in 215 unrelated individuals from Northern Bavaria. The allele frequencies obtained were: AHSG*1 = 0.7139, AHSG*2 = 0.2697 and AHSG*3 = 0.0164.

Conformational flexibility of the agonist binding jaw of the human P2X3 receptor is a prerequisite for channel opening.

BACKGROUND AND PURPOSE: It is assumed that ATP induces closure of the binding jaw of ligand-gated P2X receptors, which eventually results in the opening of the membrane channel and the flux of cations. Immobilization by cysteine mutagenesis of the binding jaw inhibited ATP-induced current responses, but did not allow discrimination between disturbances of binding, gating, subunit assembly or trafficking to the plasma membrane. EXPERIMENTAL APPROACH: A molecular model of the pain-relevant human (h)P2X3 receptor was used to identify amino acid pairs, which were located at the lips of the binding jaw and did not participate in agonist binding but strongly approached each other even in the absence of ATP. KEY RESULTS: A series of cysteine double mutant hP2X3 receptors, expressed in HEK293 cells or Xenopus laevis oocytes, exhibited depressed current responses to α,ß-methylene ATP (α,ß-meATP) due to the formation of spontaneous inter-subunit disulfide bonds. Reducing these bonds with dithiothreitol reversed the blockade of the α,ß-meATP transmembrane current. Amino-reactive fluorescence labelling of the His-tagged hP2X3 receptor and its mutants expressed in HEK293 or X. laevis oocytes demonstrated the formation of inter-subunit cross links in cysteine double mutants and, in addition, confirmed their correct trimeric assembly and cell surface expression. CONCLUSIONS AND IMPLICATIONS: In conclusion, spontaneous tightening of the binding jaw of the hP2X3 receptor by inter-subunit cross-linking of cysteine residues substituted at positions not directly involved in agonist binding inhibited agonist-evoked currents without interfering with binding, subunit assembly or trafficking.