Investigation of volatile metabolites during growth of Escherichia coli and Pseudomonas aeruginosa by needle trap-GC-MS.

A new method for the growth-dependent headspace analysis of bacterial cultures by needle trap (NT)-gas chromatography-mass spectrometry (GC-MS) was established. NTs were used for the first time as enrichment technique for volatile organic compounds (VOCs) in the headspace of laboratory cultures. Reference strains of Escherichia coli and Pseudomonas aeruginosa were grown in different liquid culture media for 48 h at 36 °C. In the course of growth, bacterial culture headspace was analysed by NT-GC-MS. In parallel, the abiotic release of volatile organic compounds (VOC) from nutrient media was investigated by the same method. By examination of microbial headspace samples in comparison with those of uninoculated media, it could be clearly differentiated between products and compounds which serve as substrates. Specific microbial metabolites were detected and quantified during the stationary growth phase. P. aeruginosa produced dimethyl sulfide (max. 125 µg L(-1) < limits of quantification (LOQ)), 1-undecene (max. 164 µg L(-1)) and 2-nonanone (max. 200 µg L(-1)), whereas E. coli produced carbon disulfide, butanal and indole (max. 149 mg L(-1)). Both organisms produced isoprene.

[Validation of a screening instrument for borderline personality disorder in adolescents and young adults - psychometric properties and association with the patient's self-esteem]. / Validierung eines Screening-Instruments zur Borderline-Persönlichkeitsstörung im Jugend- und jungen Erwachsenenalter - Gütekriterien und Zusammenhang mit dem Selbstwert der Patienten.

This investigation aimed to evaluate the German version of the BPQ as a screening instrument for borderline personality disorder (BPD) in a clinical sample. Furthermore, an association between self-esteem and BPD was examined. In a consecutive modus, 27 patients with BPD and 54 clinical controls (age range: 14 - 25 years) completed a self-report questionnaire and took part in a semi-structured interview. The German version of the BPQ revealed a high internal consistency (α = 0.95) and test-retest-reliability (r = 0.94). The criterion validity (r = 0.60) and the cut-off value (49) must be interpreted with caution due to the small sample size. BPD as well as 8 out of 9 subscales of the BPQ were significantly associated with lower self-esteem. A pre-screening using the BPQ within the clinical setting may facilitate early detection of BPD. In addition, building up self-esteem seems to be very important in the treatment of patients with BPD.

[Differential diagnosis and treatment of cheilognathopalatoschises]. / Differenzialdiagnostik und Therapie der Lippen-Kiefer-Gaumen-Spalten.

Cleft lip, alveolous and palate is the second frequent malformation in Europe with an incidence of 1 : 500. Pertaining to ontogeny it must be differentiated between cleft lip and alveolous and cleft palate. Cleft lip and cleft lip and alveolous can occur unilateral, right or left, or bilateral. Cleft bony palate can also occur unilateral, right or left, or bilateral, but cleft velum only in the median plane. Diagnostic and treatment of cleft lip and palate call for interdisciplinary cooperation between gynecologist/obstetrician, cranio-maxillo-facial surgeon, pediatrician, otorhinolaryngologist, orthodontist and logopedist. The schedule of primary cleft surgery in Germany is marked by a more-stage concept, in which at the end of the second year of life cleft lip and palate except cleft alveolous should be closed up. Despite of most careful surgery patients with cleft lip and palate can show functional and aesthetic disturbances. The functional disorders can affected masticatory function, speech, hearing and nasal breathing. Aesthetics disorders can be concerned to skeletal or soft tissue deformities of lip and nose. Operative corrections of bone and soft tissue can rehabilitate these patients entirely from functional and aesthetic view.

Incidence of T wave alternation after acute myocardial infarction and correlation with other prognostic parameters: results of a prospective study.

Tachycardia induced alternation of the T wave (TWA) has been associated with arrhythmia morbidity in mixed patient populations. However, less is known concerning the general incidence of TWA and its usefulness in risk stratification early after acute myocardial infarction (MI). TWA was prospectively and systematically assessed in 140 consecutive patients 15 +/- 6 days after acute MI and prior to discharge. Results of TWA measurements were compared to other noninvasive risk markers, LV function, and coronary angiography. Sustained TWA was present at rest or inducible during exercise in 27% of patients. The patient-specific heart rate for the onset of TWA was 98 +/- 9 beats/min. After multivariate analysis, TWA correlated with age (P = 0.02) and LV function (P = 0.002) and occurred more often in patients after nonanterior MI (P = 0.03). Acute results of Holter monitoring, late potentials by signal-averaged ECG, and heart rate variability were unrelated to the TWA status. During follow-up (451 +/- 210 days) two major arrhythmic events occurred. The incidence of TWA early after MI is about 25%. TWA is related to age and LV function but not to other common arrhythmia markers. Although TWA does not appear to be related to excessive cardiac morbidity, evaluation of the prognostic significance of TWA requires further study.

The Ras mutant D119N is both dominant negative and activated.

The introduction of mutation D119N (or its homolog) in the NKxD nucleotide binding motif of various Ras-like proteins produces constitutively activated or dominant-negative effects, depending on the system and assay. Here we show that Ras(D119N) has an inhibitory effect at a cell-specific concentration in PC12 and NIH 3T3 cells. Biochemical data strongly suggest that the predominant effect of mutation D119N in Ras-a strong decrease in nucleotide affinity-enables this mutant (i) to sequester its guanine nucleotide exchange factor, as well as (ii) to rapidly bind GTP, independent of the regulatory action of the exchange factor. Since mutation D119N does not affect the interaction between Ras and effector molecules, the latter effect causes Ras(D119N) to act as an activated Ras protein at concentrations higher than that of the exchange factor. In comparison, Ras(S17N), which also shows a strongly decreased nucleotide affinity, does not bind to effector molecules. These results point to two important prerequisites of dominant-negative Ras mutants: an increased relative affinity of the mutated Ras for the exchange factor over that for the nucleotide and an inability to interact with the effector or effectors. Remarkably, the introduction of a second, partial-loss-of-function, mutation turns Ras(D119N) into a strong dominant-negative mutant even at high concentrations, as demonstrated by the inhibitory effects of Ras(E37G/D119N) on nerve growth factor-mediated neurite outgrowth in PC12 cells and Ras(T35S/D119N) on fetal calf serum-mediated DNA synthesis in NIH 3T3 cells. Interpretations of these results are discussed.

[Augmentation of the extremely atrophied maxilla and mandible by autologous calvarial bone transplantation]. / Aufbau des extrem atrophierten Ober- und Unterkiefers durch autologe Kalottenknochentransplantate.

Rehabilitation in patients with severe alveolar ridge atrophy of the maxilla or mandible is problematic and can often only be achieved by long-term treatment. In most cases, autologous bone grafting with iliac crest bone has been used to augment severely atrophied upper jaws. In our experience, iliac bone grafts are less useful, since iliac bone appears to be of inferior quality; in elderly osteoporotic women, the bone is soft, indentable, and of poor osteogenic potency. In our department, we have been using only autologous calvarial bone grafts for augmentation of alveolar ridge atrophy since 1993. The bone is removed from the outer table of the skull only, trimmed to the alveolar ridge, und fixed with titanium lag scews. The skull defect created is covered with crushed bone or a titanium mesh to avoid aesthetic problems. Insertion of dental implants follows after a healing period of the bone grafts of 5-6 months. A total of 63 patients underwent calvarial split-graft augmentation; augmentation of the maxilla and mandible was carried out in 15 of these patients, of the maxilla only in eight, and of the mandible only in 40. The investigations 1 year later showed a resorption rate of approximately 10%. This is lower than when using iliac bone grafting. The resorption results were stable between 6 and 12 months after augmentation. Using dental implants (12 patients with 32 implants), the resorption rate was low and constant. We have never seen total loss of bone grafts or intracranial complications. All patients were pleased with the treatment. In our opinion, severe alveolar atrophy of the maxilla or mandible should be compensated for by augmentation with autologous calvarial bone grafts to obtain good long-term results.

[Significance of hemodynamic parameters of blood loss in orthognathic surgery]. / Bedeutung hämodynamischer Parameter für den Blutverlust in der Dysgnathiechirurgie.

INTRODUCTION: The hemodynamic parameters of 95 patients undergoing maxillary or bimaxillary orthognathic surgery in 1996 and 1997 at the Department of OMF Surgery/Plastic Surgery, Krefeld, Germany, were analyzed retrospectivly to study the effect of intraoperative blood loss. MATERIALS AND METHODS: The parameters included the blood loss volume, age, weight and sex of the patients, the mode of osteotomy and the operation time, the surgeon, the average blood pressure, the infusion volume, the anesthesiologist, the thrombocyte counts and their function, the activity of the coagulation factors II, V, VII, VIII, IX, X, XI, XII, XIII and von-Willebrand-factor, and the pathological coagulation factor counts of each patient, the rate of autologous blood donation and the rate of retransfusion. Statistical analysis was done using the Speraman-Rhotest. RESULTS: The average blood loss during maxillary osteotomy was 670 +/- 380 ml and during bimaxillary surgery 1120 +/- 510 ml. Men lost about 300 ml more than women. Operations of more than 3.5 h in length led to a blood loss of 1200 +/- 520 ml as opposed to 670 +/- 310 ml. The average blood loss among various surgeons was between 670 ml and 1180 ml of various anesthesiologists between 730 ml and 1200 ml, without statistical evidence. Some 17.9% of patients showed pathological thrombocytic function concerning medication with aspirin; 34.7% had pathological activities of coagulation factors, but only 2.1% with clinical significance. CONCLUSION: Mode of operation, maxillary or bimaxillary, und length of operation were the most significant factors of intraoperative blood loss. Patients with pathological coagulation had nearly the same rate of blood loss as patients with physiological coagulation. In most cases this was determined by restriction of aspirin. Analysis of the rate of autologous blood retransfusion showed a significant correlation to blood loss in bimaxillary surgery. Maxillary osteotomy led to a retransfusion of only 14.2% of autologous blood unit. This should be reviewed critically especially concerning costs.

Crystal structure of the hexamerization domain of N-ethylmaleimide-sensitive fusion protein.

N-ethylmaleimide-sensitive fusion protein (NSF) is a cytosolic ATPase required for many intracellular vesicle fusion reactions. NSF consists of an amino-terminal region that interacts with other components of the vesicle trafficking machinery, followed by two homologous ATP-binding cassettes, designated D1 and D2, that possess essential ATPase and hexamerization activities, respectively. The crystal structure of D2 bound to Mg2+-AMPPNP has been determined at 1.75 A resolution. The structure consists of a nucleotide-binding and a helical domain, and it is unexpectedly similar to the first two domains of the clamp-loading subunit delta' of E. coli DNA polymerase III. The structure suggests several regions responsible for coupling of ATP hydrolysis to structural changes in full-length NSF.

Kinetic analysis by fluorescence of the interaction between Ras and the catalytic domain of the guanine nucleotide exchange factor Cdc25Mm.

Guanine nucleotide exchange factors (GEFs) activate Ras proteins by stimulating the exchange of GTP for GDP in a multistep mechanism which involves binary and ternary complexes between Ras, guanine nucleotide, and GEF. We present fluorescence measurements to define the kinetic constants that characterize the interactions between Ras, GEF, and nucleotides, similar to the characterization of the action of RCC1 on Ran [Klebe et al. (1995) Biochemistry 34, 12543-12552]. The dissociation constant for the binary complex between nucleotide-free Ras and the catalytic domain of mouse Cdc25, Cdc25(Mm285), was 4.6 nM, i.e., a 500-fold lower affinity than the Ras.GDP interaction. The affinities defining the ternary complex Ras. nucleotide.Cdc25(Mm285) are several orders of magnitude lower. The maximum acceleration by Cdc25(Mm285) of the GDP dissociation from Ras was more than 10(5)-fold. Kinetic measurements of the association of nucleotide to nucleotide-free Ras and to the binary complex Ras. Cdc25(Mm285) show that these reactions are practically identical: a fast binding step is followed by a reaction of the first order which becomes rate limiting at high nucleotide concentrations. The second reaction is thought to be a conformational change from a low- to a high-affinity nucleotide binding conformation in Ras. Taking into consideration all experimental data, the reverse isomerization reaction from a high- to a low-affinity binding conformation in the ternary complex Ras. GDP.Cdc25(Mm285) is postulated to be the rate-limiting step of the GEF-catalyzed exchange. Furthermore, we demonstrate that the disruption of the Mg2+-binding site is not the only factor in the mechanism of GEF-catalyzed nucleotide exchange on Ras.

Biochemical and biological consequences of changing the specificity of p21ras from guanosine to xanthosine nucleotides.

The D119N mutation of p21ras was prepared by site-directed mutagenesis. Its nucleotide binding properties were investigated using fluorescently labelled guanosine and xanthosine nucleotides. Its affinity for guanosine nucleotides is severely reduced, with a concomitant increase in the affinity for xanthosine nucleotides, which leads to an almost complete reversal of base specificity. The protein is a GTPase as well as a XTPase and the hydrolysis reaction can be efficiently stimulated by GAP. Dissociation of XDP from the mutant is stimulated by the guanine nucleotide exchange factor Cdc25Mm in a similar manner to that of GDP from wildtype. The interaction of the mutant with the effector domain of c-Raf kinase or Ral-GEF is normal. In microinjection experiments in PC12 and NIH3T3 cells the protein behaves as an oncogenic mutant due to its high dissociation rate for GDP. However, when the protein is loaded with XDP before microinjection the onset of the oncogenic signal can be efficiently retarded. Thus, the protein behaves initially as wildtype and later as an oncogenic protein.

Modulation of ryanodine-induced contractures in human skeletal muscle pretreated with dantrolene.

Dantrolene seems to be the causal therapy in malignant hyperthermia (MH) crisis but the complex mechanisms of MH and dantrolene therapy are still not fully understood. The influence of dantrolene on ryanodine-induced contractures has been reported in animal studies only. In the present study 20 patients from 17 families were tested for MH using the protocol of the European Malignant Hyperthermia Group. In addition ryanodine-induced contractures were evaluated following bolus application of 10.0 mumol.l-1 ryanodine. After pretreatment with 1 mumol.l-1 dantrolene ryanodine-provoked contractures developed significantly later in MHS (15.8 +/- 1.8 min) and MHN (46.0 +/- 4.2 min) muscle specimens than after ryanodine alone (MHS 4.8 +/- 0.7 min. (MHN 13.7 +/- 0.9 min). They were no longer observed in either group after pretreatment with 5 mumol.l-1 dantrolene. We conclude that dantrolene is able to attenuate ryanodine-induced contractures dose-dependently, and therefore it is speculated that dantrolene could specifically act at the ryanodine receptor binding site.

C1840-T mutation in the human skeletal muscle ryanodine receptor gene: frequency in northern German families susceptible to malignant hyperthermia and the relationship to in vitro contracture response.

In swine, a point mutation in the ryanodine receptor gene can account for all cases of malignant hyperthermia (MH). The frequency of a corresponding mutation in humans (C1840-T) and its relationship to the in vitro contracture profile is unknown. We screened 192 patients from 28 unrelated northern German families for the C1840-T mutation in the human ryanodine receptor gene and tested for MH susceptibility using the in vitro contracture test (IVCT) according to the European MH Protocol. In our patients 106 revealed MH susceptible (MHS), 56 MH nonsusceptible and 30 MH equivocal status following IVCT. In each family one or two individuals had developed clinical signs of MH or a MH crisis. All of these patients were classified MHS. The C1840-T mutation was found in 2 of 28 families (7.1%). All eight individuals of the two families characterized by this mutation revealed MHS status following IVCT. The thresholds for halothane- and caffeine-induced contractures as well as the contracture profiles following cumulative (0.4-10.0 mumol/l every 3 min) and bolus (10 mumol/l) administration of ryanodine were found to be similar in MHS patients with and without the C1840-T mutation. In conclusion, the C1840-T mutation in the human ryanodine receptor gene is a rare abnormality in MHS families. Similar contracture profiles in the presence and absence of this mutation might imply no major functional role with respect to the contracture response. At present, molecular genetic analysis cannot replace IVCT to discover MH susceptibility in humans.

High-purity ryanodine and 9,21-dehydroryanodine for in vitro diagnosis of malignant hyperthermia in man.

Susceptibility to malignant hyperthermia (MH) is currently diagnosed by the in vitro contracture test (IVCT) in skeletal muscle. However, this test does not possess absolute specificity. Thus, in addition to the established procedure, the "ryanodine contracture test" has been proposed to improve discrimination between MH-susceptible (MHS) and normal (MHN) patients. In all previous studies, the ryanodine used was a mixture consisting of high-purity ryanodine (HPR) and 9,21-dehydroryanodine (DHR). Therefore, in this study the effects of both substances were investigated in concentrations of 2, 5 and 10 mumol litre-1. With all concentrations, contractures appeared earlier in MHS than in MHN muscles, but these differences were significant at all contracture levels with HPR only. Moreover, with the smallest concentration (2 mumol litre-1), the best discrimination between MHS and MHN was observed. Classification of MH-equivocal patients (MHE) as MHS or MHN seems to be possible with the use of ryanodine-induced contractures. The contracture test with HPR should therefore be added to the established procedure of the IVCT.

[High purity ryanodine for in vitro diagnosis of malignant hyperthermia]. / Hochreines Ryanodin zur In-vitro-Diagnostik der malignen Hyperthermie.

OBJECTIVE AND STUDY DESIGN: The identification of disposition for malignant hyperthermia (MH) is performed by the halothane-caffeine contracture test in skeletal muscle. However, testing currently renders about 14% of the patients MH equivocal (MHE). To reduce this number the "ryanodine contracture test" has been proposed in addition to the established procedure. As all ryanodine preparations used until now are combinations of ryanodine and dehydroryanodine, we investigate in this study the effects of the now available high-purity ryanodine (HPR) in concentrations of 2,5 and 10 mumol/l. RESULTS: With all concentrations contractures appeared significantly earlier in MHS (malignant hyperthermia susceptible) than in MHN (malignant hyperthermia non-susceptible). However, only at 2 mumol/l a time overlap between both groups was not observed. By defining a time interval for MHS and MHN, three of the MHE patients could be assigned to MHS and three to MHN following their ryanodine-induced contractures. CONCLUSION: The ryanodine contracture test should be added to the current diagnostics of malignant hyperthermia.

Accelerated contractures after administration of ryanodine to skeletal muscle of malignant hyperthermia susceptible patients.

A genetic disorder of the calcium releasing ryanodine receptor has recently been postulated in malignant hyperthermia (MH) and ryanodine-induced contractures differ between subjects who are malignant hyperthermia susceptible (MHS) and non-susceptible (MHN). We tested 39 patients from 26 families for MH, using the procedure of the European Malignant Hyperthermia Group. A ryanodine contracture test was performed by both cumulative (0.4-10.0 mumol litre-1 every 3 min) and bolus (10.0 mumol litre-1) application. Contracture with cumulative ryanodine application started significantly earlier in MHS (9.6 (SEM 0.5) min) than in MHN patients (24.6 (1.3) min). A significant difference in start of contracture between MHS (4.8 (0.6) min) and MHN (14.5 (0.6) min) patients occurred also after bolus application of ryanodine. The ryanodine contracture test seems to be a potentially specific in vitro diagnostic test for MH.

[Ryanodine-induced contractures for the diagnosis of malignant hyperthermia susceptibility]. / Ryanodin-induzierte Kontrakturen zur Diagnose der Maligne-Hyperthermie-Veranlagung.

The halothane-caffeine contracture test is presently the most well-established method for identification of malignant hyperthermia susceptibility (MHS) or non-susceptibility (MHN). However, 10-20% of the patients tested are classified as equivocal (MHE), i.e. their susceptibility remains uncertain. A genetic disorder of the calcium releasing ryanodine receptor has been postulated recently. Therefore, 12 patients were tested in addition to the protocol of the European Malignant Hyperthermia Group (EMHG) for dose- and time-dependent contracture after ryanodine application. In this study, contracture of 0.2g appeared significantly earlier in MHS patients (17.5 +/- 1.7 min; n = 5) during cumulative ryanodine exposition (0.4-0.8-1.6-10.0 mumol/l) than in MHN (38.2 +/- 5.4 min; n = 5). A significant difference between MHS (10.0 +/- 1.7 min; n = 6) and MHN (19.8 +/- 0.6 min; n = 3) was also seen after bolus application of ryanodine (10.0 mumol/l). One patient classified as MHE according to the EMHG protocol, manifested as MHN after the ryanodine contracture test. This study supports previous work suggesting the ryanodine contracture test as an improvement in the in-vitro diagnosis of MH susceptibility.

Characterisation of glutamine uptake in rat liver mitochondria.

Glutamine is taken up by rat liver mitochondria in an electroneutral manner with a Km of 3.3 mM and a Vmax of 33 nmol x min-1 x mg-1 at 10 degrees C. The uptake is driven by the mitochondrial pH/cytosolic pH difference in isolated mitochondria, as well as in the intact rat liver. The rate of uptake is stimulated at a more alkaline matrix pH due to a stimulation of mitochondrial glutaminase. Our data confirm the notion that glutamine metabolism is regulated by pH, not only at the site of its metabolism but also through regulation of its transport systems.