Reevaluation of citrate lyase from Escherichia coli.

The subunit structure of citrate lyase from Escherichia coli was shown to be similar to that of all other lyases investigated so far. The three different subunits with molecular masses of 55.5 kDa, (large subunit) 35 kDa (medium-sized subunit) and 12.5 kDa (small subunit, acyl carrier protein) occurred in a ratio of 1:1:1. Using high-pressure liquid chromatography, it was possible to demonstrate that the reported large acyl carrier protein, with a molecular mass of 85 kDa was a contaminating protein associated with citrate lyase multienzyme complex; it could be removed by anion-exchange chromatography with Q-Sepharose. The typical two configurations of citrate lyase, the 'star' form and the 'ring' form with a diameter of 14.3 nm and 15.4 nm, respectively, could be detected by electron microscopy.

The cysteine residue of the SoxY protein as the active site of protein-bound sulfur oxidation of Paracoccus pantotrophus GB17.

Four proteins of Paracoccus pantotrophus are required for hydrogen sulfide-, sulfur-, thiosulfate- and sulfite-dependent horse heart cytochrome c reduction. The lack of free intermediates suggested a protein-bound sulfur oxidation mechanism. The SoxY protein has a novel motif containing a cysteine residue. Electrospray ionization and matrix-assisted laser desorption ionization mass spectrometry of the SoxYZ protein revealed one mass for SoxZ and different masses for SoxY, indicating native SoxY (10977 Da) and SoxY with additional masses of +32, +80, +112 and +144 Da, suggesting addition of sulfur, sulfite, thiosulfate and thioperoxomonosulfate. Reduction of SoxY removed the additional masses, indicating a thioether or thioester bond. N-Ethylmaleimide inhibited thiosulfate-oxidation and the kinetics suggested a turn-over-dependent mode of action. These data were evidence that the sulfur atom to be oxidized was covalently linked to the thiol moiety of the cysteine residue of SoxY and the active site of sulfur oxidation.

Re-operation for recurrent colorectal cancer: the importance of early diagnosis for resectability and survival.

Between 1978 and 1986, 179 patients with recurrent colorectal cancer were treated and 137 patients were operated a second time.82.1% of the patients showed elevated CEA levels (greater than or equal to 5 ng/ml) at the time of diagnosis. In 58.1% of the patients the CEA increase preceded the recognition of recurrence, and in 13.4% the diagnosis could be confirmed only by a second-look operation. In 46.7% of the re-operated patients a potentially curative resection of the locally recurrent or metastatic disease could be performed. The resectability was significantly lower in patients with symptomatic recurrent disease (34.5%) as compared to asymptomatic patients with CEA-directed positive imaging (52.7%) and the second-look patients (62.5%) respectively. A significant improvement in survival could be achieved especially in the second-look operated patients.

Evaluation of Ca 12-5 as a tumor marker for gastric and colo-rectal cancer in comparison to CEA and Ca 19-9.

We performed simultaneous measurements of CA 12-5, CEA and Ca 19-9 in the preoperative sera of 87 patients with gastric cancer, 177 patients with colo-rectal cancer and 55 patients with benign diseases. 5.6% of the control patients, 13.8% of the gastric cancer patients and 9.6% of the colo-rectal cancer patients showed Ca 12-5 values of 27.0 U/ml and more. In contrast, the sensitivity of the tumor markers CEA (greater than or equal to 5.0 ng/ml in 17.1% of the gastric cancer and 36.2% of the colo-rectal cancer patients examined) and Ca 19-9 (greater than or equal to 25.0 U/ml in 16.1% of the gastric cancer and 19.8% of the colo-rectal cancer patients) was distinctly higher. The simultaneous determination of three markers on the one hand increased the rate of 'marker-positive' patients (up to 34.5% in gastric cancer and 45.2% in colo-rectal cancer) but on the other hand diminished the specificity (from 96.4% to 89.1%) of the examination.

Comparative investigation on copper oxides by depth profiling using XPS, RBS and GDOES.

Depth profiling has been performed by using X-ray photoelectron spectrometry (XPS) in combination with Ar-ion sputtering, Rutherford backscattering spectrometry (RBS) and glow discharge optical emission spectrometry (GDOES). The data obtained by XPS have been subjected to factor analysis in order to determine the compositional layering of the copper oxides. This leads to two or three relevant components within the oxide layers consisting of Cu(2)O or CuO dependent on the sample preparation. GDOES measurements show sputtering profiles which are seriously influenced by a varying sputter rate. To ensure the results obtained so far, RBS measurements of the oxide layers have been carried out in order to discover artefacts of the other methods used and to demonstrate the excellent suitability of RBS for quantitative analysis of these layers. Chemical analysis consisting of (1) carrier-gas fusion analysis (CGFA) and (2) selective dissolution of Cu(2)O/CuO allows the determination of the total amount of oxygen and copper, respectively, and can serve as a cornerstone of quantitative analysis.

Cystic lymphangioma of the small-bowel mesentery: case report and a review of the literature.

Cystic lymphangioma of the small-bowel mesentery is a rare manifestation of an intraabdominal tumor in elderly patients. We present a case of a small-bowel mesentery lymphangioma, causing fever and chills and present clinical and pathologic features. Furthermore, etiology and differential diagnosis of this tumor are discussed.

[Key role of the CEA test in the diagnosis and surgical therapy of recurrent colorectal cancer]. / Schlüsselrolle des CEA-Testes für die Diagnostik und chirurgische Therapie des rezidivierten colorectalen Carcinoms.

Between 1978 and 1984, 87 patients with recurrent colorectal cancer have been operated upon. In 10 of 35 patients with locoregional recurrence and 24 of 52 with distant metastases therapy was potentially curative. Of 87 patients 73 had elevated CEA levels (greater than or equal to 5 ng/ml) at the time of diagnosis. In 65 of 73 patients the CEA increase preceded the recognition of recurrence and in 14 patients the diagnosis could be confirmed only by a second-look operation. Patients with metastases (91.3%) showed CEA elevation more often than those with locoregional recurrence (71.4%). Patients with operable disease had significantly (p less than 0.05) lower CEA values (median 19.7 ng/ml) than those with inoperable recurrent carcinomas (median 36.9 ng/ml).

[Measurement of uranium isotope ratio in solid sample by laser ablation and double-beam diode laser atomic absorption].

The 235U/238U isotope ratio was determined by diode laser atomic absorption spectrometry in a plasma induced by a Nd:YAG laser. Two diode lasers, which probed almost the same plasma volume, were used as light sources. 235U and 238U absorption signals were simultaneously measured by tuning the diode lasers to the absorption lines 682.6736 nm and 682.0768 nm, respectively. The optimal focus position for the Nd:YAG laser was found to be 0.4 cm below the sample surface. The precision and accuracy of 235U/238U isotope ratio determination were obtained to be 5% and 2%, respectively. The detection limit of the 235U isotope, evaluated on the basis of 3sigma criteria, was estimated to be 18 microg x g(-1). All these results show an improvement compared with the method in which only one diode laser was used as a light source.

[Cerclage using hollow screws for internal arthrodesis in cases of pronounced osteoporosis and osteopenia]. / Hohlschraubenunterstützte Rahmencerclage zur internen Arthrodese bei ausgeprägter Osteoporose und -penie.

Osteosynthesis of osteoporotic or osteopenic bone poses a considerable challenge for the surgeon, the type of osteosynthesis performed, and the osteosynthesis material employed. In osteoporotic and osteopenic bone, especially when circulation is decreased or sensitivity reduced, the principle of tension banding places great demands on the bone and surgeon since insufficient tightening of the wire results in inadequate immobilization while overtightening of the tension band can cause the wire to cut through the soft bone. This contribution describes a surgical technique, which prevents the wire from cutting through the bone. Cannulated screws are inserted vertically into the bone in the traction direction and the wire of the tension banding is passed through the cannulated screws. This ensures that the wire traction is distributed across the entire cross section of the bone. Thus, it is possible to completely tighten the tension band in osteopenic and osteoporotic bone. This technique is illustrated using the example of internal screw and tension band arthrodesis of the ankle joint, which achieved stable arthrodesis with weight bearing and recovered ambulation in a patient with pronounced diabetic foot syndrome and peripheral arterial occlusive disease.

Characterization of citrate lyase from Clostridium sporosphaeroides.

Cells of Clostridium sporosphaeroides which were grown on citrate contained citrate lyase and citrate lyase acetylating enzyme, but no detectable citrate synthase and citrate lyase deacetylase activities. Citrate lyase from C. sporosphaeroides was purified to homogeneity as judged by polyacrylamide gel electrophoresis and high performance liquid chromatography. In contrast to the enzyme from Clostridium sphenoides, the addition of L-glutamate was not necessary for activity and stabilization of the enzyme. The purified enzyme had a specific activity of 34 U/mg protein and was comparable to other citrate lyases with respect to its molecular weight and subunit composition. Electron microscopic investigations showed that similar to the lyase from C. sphenoides and in contrast to all other citrate lyases examined so far, the majority of the enzyme molecules was present in "star" form.

Transfer and expression of degradative and antibiotic resistance plasmids in acidophilic bacteria.

The genetic accessibility of selected acidophilic bacteria was investigated to evaluate their applicability to degrading pollutants in acidic environments. The IncP1 antibiotic resistance plasmids RP4 and pVK101 and the phenol degradation-encoding plasmid pPGH11 were transferred from neutrophilic bacteria into the extreme acidophilic eubacterium Acidiphilium cryptum at frequencies of 1.8 x 10(-2) to 9.8 x 10(-4) transconjugants per recipient cell. The IncQ antibiotic resistance plasmid pSUP106 was mobilizable to A. cryptum by triparental matings at a frequency of 10(-5) transconjugants per recipient cell. In the transconjugants, antibiotic resistances and the ability to degrade phenol were expressed. A. cryptum AC6 (pPGH11) grew with 2.5 mM phenol at a doubling time of 12 h and a yield of 0.52 g (dry cell weight) per g of phenol. A. cryptum harbored five native plasmids of 255 to 6.3 kb in size. Plasmids RP4 and pVK101 were transferred from Escherichia coli into Acidobacterium capsulatum at frequencies of 10(-3) and 2.3 x 10(-4) and to the facultative autotroph Thiobacillus acidophilus at frequencies of 1.1 x 10(-5) and 2.9 x 10(-6) transconjugants per recipient cell, respectively. Plasmid pPGH11 could not be transferred into the latter strains. T. acidophilus wild type contained six so far cryptic plasmids of 220 to 5 kb.

Attenuation of insulin actions in primary rat hepatocyte cultures by phenylarsine oxide.

Phenylarsine oxide (PAO), a trivalent arsenical which complexes vicinal dithiols, prevented the action of insulin in primary cultured adult rat hepatocytes. Simultaneous short-term treatment of 48-h old cells with insulin and 2 microM PAO resulted in complete attenuation of the insulin-dependent increase in the level of fructose 2,6-bisphosphate and the activation of phosphofructokinase 2, pyruvate kinase, glucokinase flux and glycolysis. Basal rates of glucose transport and glycolysis were not affected. PAO also abolished stimulation of glycogen synthesis and amino-acid transport and the decrease of glycogenolysis evoked by insulin. The 20-fold activation of the insulin receptor tyrosine kinase by insulin was, however, not reduced by PAO. The data suggest that in differentiated hepatocytes insulin signal transduction involves vicinal sulhydryls located at a post-receptor step.

Evaluation of the secretory component as a prognostic variable in colorectal carcinoma.

Expression of the secretory component (SC) of IgA was investigated in normal colonic mucosa, in adenomas and in 188 colon carcinomas. Of the carcinoma patients, 149 underwent potentially curative surgical treatment. In particular, it was of interest to determine whether pathohistological or clinical parameters showed any association with the mode of SC expression in carcinomas. Irrespective of anatomical site, normal colon mucosa was generally SC-positive. Normal epithelium, however, displayed microheterogeneity in SC content, which was related to columnar and goblet-cell type and microtopographical site, implying a subtle regulatory mechanism. In contrast to adenomas, which were evenly SC-positive and showed focally weak SC expression in only 2 out of 20 cases, carcinomas were rather heterogeneous in their SC-expression pattern. In all, 48 carcinomas were entirely positive, 48 were completely negative and the remaining 92 showed various patterns of heterogeneous SC expression. The mode of SC expression was correlated with tumor staging, i.e., complete absence of SC was more often found in Dukes' C and D groups. In addition, the presence of SC in carcinomas was statistically correlated with the mucinous type. The mode of SC expression was not correlated with tumor grading and tumor location. The rate of disease-free survival and the risk of tumor-related death revealed a significant correlation with the mode of SC expression when patients who had received potentially curative or non-curative treatment were included in the log-rank test. Absence or low levels of SC expression were more frequent in the group that suffered a tumor relapse than in the recurrence-free cohort. Tumor-related death was more frequent when primaries were characterized by absence or low levels of SC expression. We conclude that absence or low levels of SC expression are an unfavorable prognostic variable in colorectal carcinoma.

Differential expression of beta 1, beta 3, and beta 4 integrin subunits in nonneoplastic neural cells of the peripheral and autonomic nervous system and in tumors derived from these cells.

BACKGROUND: Extracellular matrix proteins and their receptors take part in physiologic neural development and organization and also in abnormal neoplastic growth and spread. There is increasing evidence for the implication of integrins in these processes. EXPERIMENTAL DESIGN: Human tissues containing nonneoplastic neural cells of the peripheral and autonomic nervous system and a comprehensive series of neural tumors were examined for the in situ expression of beta 1, beta 3, and beta 4 integrins. Serial frozen sections of each tissue sample were immunostained using an indirect streptavidin/biotin-peroxidase method and monoclonal antibodies against beta 1, alpha 1 to alpha 6, beta 3, alpha v, and beta 4 subunits. RESULTS: Both small- and large-diameter nerve fibers of normal peripheral nerve trunks were consistently beta 1+, alpha 6+, and beta 4+ in the absence of alpha 3, alpha 4, alpha 5, and beta 3. Small-diameter nerve fibers further expressed alpha 1, alpha 2, and alpha v. Meissner's corpuscles and inner cores of Pacinian corpuscles shared the integrin repertoire of small-diameter nerve fibers with additional expression of alpha 3; outer cores of Pacinian corpuscles were beta 1+, alpha 3+, alpha 6+, and beta 4+. Regenerating nerve fibers paralleled the integrin profile of normal peripheral nerves. By contrast, malignant schwannomas showed considerable changes in integrin expression. These alterations consisted mainly in a neoexpression of alpha 3, alpha 4, and alpha 5 and in an abnormal loss of alpha 6 and beta 4. Expression of alpha 1, alpha 2, and alpha v was variable; absence of beta 3 was generally conserved. In ganglion cells, integrin expression was restricted to beta 1 and alpha 3 subunits, and chromaffine cells of the adrenal medulla even lacked any detectable beta 1, beta 3, and beta 4 integrin subunits. (Ganglio)-neuroblastomas, however, were beta 1+, alpha 1+, and alpha 3+, whereas primitive peripheral neuroectodermal tumors were beta 1+ and alpha 5+. CONCLUSIONS: Nonneoplastic human neural cells exhibit a complex and, at the same time, differential pattern of beta 1, beta 3, and beta 4 integrin subunit expression. Malignant transformation leads to considerable changes in this integrin profile. The observation of neoplasia-associated abnormalities underlines the important role of integrins in the orderly development and maintenance of human neural tissue. Some aspects of the emerging integrin subunit patterns are useful for the differential diagnosis of neural soft-tissue tumors.

The classic nail in the therapy of trochanteric fractures.A prospective, controlled study.

In a prospective controlled study we analysed the classic nail, a new intramedullary implant for the fixation of peritrochanteric fractures. By means of lateral bending of 4 degrees, unreamed implantation is generally possible. Our collective of 85 patients was characterized by elderly mean age (82.3 years), a predominance of female patients (4.3 : 1), a minor trauma aetiology without relevant additional injuries, and a high rate of concomitant disease (92%). Only 31A-type femur fractures were treated, with a low specific complication rate of 13%, whereby operative revision was necessary in only two patients (3%). A good anatomic reconstruction and full weight-bearing with the osteosynthesis was achieved in the majority of patients. No secondary shaft fractures and only one case of cutting out was observed. The mortality (30 days: 18%, 6 months: 25%) as well as the high rate of unspecific complications (20%) were caused by the patients' multiple morbidity. Deficits in the Merle d'Aubigne score at follow-up after 6 months can be interpreted within the same context. Nevertheless, 85% could return to their former social environment and only 15% became dependent on a nursing institution in connection with the fracture treatment.

[The Richard classic nail (intramedullary hip screw, IMHS) as unreamed intramedullary nail in osteosynthesis treatment of pertrochanteric fractures]. / Der Classic Nagel nach Richards (Intramedullary Hip Screw, IMHS) als unaufgebohrter Marknagel bei der osteosynthetischen Versorgung pertrochanterer Frakturen.

The treatment of 85 patients with pertrochanteric femoral fractures (average age 82.3 years) with the Richards classic nail allowed full weight-bearing in 94.1% and unreamed implantation in 91.8%. In the follow-up period (11.2 months post-operatively) few complications (1.2% infections, 1.2% cutting out) could be documented. The Sanders and Regazzoni score revealed that 66% of the patients reached their preoperative status again. Statistical analysis showed a negative influence of patient age and fracture type on the postoperative walking ability.

Differential expression of beta 1 integrins in nonneoplastic smooth and striated muscle cells and in tumors derived from these cells.

Integrins are a superfamily of transmembrane alpha beta heterodimers that play an important role in cell-matrix and cell-cell interactions by acting as receptors for extracellular matrix proteins and for cell adhesion molecules. Using monoclonal antibodies against beta 1, alpha 1 to alpha 6, and alpha v subunits, the in situ distribution pattern of beta 1 integrins was examined immunohistochemically in nonneoplastic smooth and striated muscle cells and in their tumors. Nonneoplastic smooth muscle cells were beta 1+, alpha 1+, alpha 3+, alpha v+ and, in diverse localizations, also alpha 5+ or even alpha 6+. The expression of the beta 1 chain was conserved in all leiomyomas and leiomyosarcomas. The distribution pattern of the alpha subunits by contrast underwent several changes during malignant transformation of smooth muscle cells. These alterations consisted in a neoexpression of alpha 2, alpha 4, and alpha 6 as well as in an abnormal abrogation of alpha 1 and alpha 3 in some leiomyosarcomas. Except for the absence of alpha 5 in the majority of epithelioid leiomyosarcomas, expression of the alpha 5 and alpha v subunits was mainly conserved. In addition, tumors with epithelioid differentiation differed from typical cases by the absence of alpha 1 and the simultaneous presence of alpha 4. Adult striated muscle cells were beta 1+ but alpha 1- to alpha 6- and alpha v-, whereas fetal striated muscle cells were not only beta 1+ but also alpha 3+/-, alpha 4+/-, alpha 5+ and alpha 6+. In all rhabdomyosarcomas the expression of beta 1 was retained. Furthermore, the majority of cases showed the expression of one or more alpha subunits most of which, ie, alpha 4, alpha 5, and alpha 6, were also found in fetal striated muscle cells. In conclusion, beta 1 integrins exhibited a differential expression pattern along the two lines of myogenic differentiation. This integrin profile underwent characteristic changes during malignant transformation. Nevertheless, the compiled distribution patterns of the alpha 1, alpha 3, and alpha v subunits allowed in most instances the discrimination between tumors of smooth (alpha 1+/alpha 3+/alpha v+) and striated muscle (alpha 1-/alpha 3-/alpha v-) differentiation.

Stimulation by insulin of glycolysis in cultured hepatocytes is attenuated by extracellular ATP and puromycin through purine-dependent inhibition of phosphofructokinase 2 activation.

Activation of glycolysis by insulin in cultured rat hepatocytes is preceded by an activation of phosphofructokinase 2 (PFK 2) and subsequent rise of the fructose 2,6-bisphosphate [Fru(2,6)P2] level. Extracellular addition of ATP or puromycin prevented the hormonal effect on glycolysis. The mechanism through which the purines abolished glycolytic stimulation was investigated. 1. 50 microM ATP completely prevented the 3-5-fold insulin-dependent increase of glycolysis, irrespective of whether the cells initially possessed a low or a high Fru(2,6)P2 content. 50 microM puromycin prevented the stimulation of glycolysis by insulin only in cells whose initial Fru(2,6)P2 levels were low and had to be increased by insulin prior to the increase in glycolysis. It did not antagonize the action of insulin cells with initial high Fru(2,6)P2 content. 2. ATP exerted effects on its own; it decreased initially high Fru(2,6)P2 levels by 95% within 10 min and decreased the basal glycolytic rate by 60%. Half-maximal effects on the Fru(2,6)P2 level were obtained with about 25 microM ATP or 15 microM adenosine 5'[beta, gamma-methylene]triphosphate. ADP and adenosine-5-[gamma-thio]triphosphate were as effective as ATP, whereas 100 microM adenosine 5'[alpha, beta-methylene]triphosphate elicited no effect. Puromycin neither decreased high Fru(2,6)P2 levels nor inhibited basal glycolysis. 3. Extracellular ATP (100 microM) led to inhibition of the active form of PFK 2. Intracellular levels of Glc6P, citrate, ATP, ADP and AMP were increased by extracellular ATP, the phosphoenolpyruvate content was decreased, Fru6P and glycerol 3-phosphate levels stayed constant. Puromycin did not inhibit PFK 2. 4. Both puromycin and ATP prevented the insulin-dependent rise of the Fru(2,6)P2 level, they abolished the activation of PFK 2 by the hormone. Puromycin did not block the accumulation of Fru(2,6)P2 provoked by glucose addition; ATP also antagonized the glucose-dependent increase. 5. 100 microM ATP elevated the cAMP-dependent protein kinase activity ratio from 0.1 to 0.38 and increased the level of inositol trisphosphate by 16-fold within 5 min, whereas puromycin was without effect on either level. It is concluded that the two purines block the insulin effect on glycolysis by preventing the hormone increasing the Fru(2,6)P2 level. The mode of action, however, seems to be different: ATP antagonizes insulin action in that it leads to increased inhibition of PFK 2 whereas puromycin prevents the activation of PFK 2 by insulin.

[Methodology, technical prerequisites and postoperative morbidity of intraoperative radiotherapy (IORT) of soft tissue sarcomas. Heidelberg Krankengut 6/91-9/92]. / Methodik, technische Voraussetzungen und postoperative Morbidität der intraoperativen Radiotherapie (IORT) bei Weichteilsarkomen. Heidelberger Krankengut 6/91-9/92.

Since June 1991 the IORT facility has operated a dedicated linear accelerator, which was installed within the central operating theater of the Department of Surgery. As of 9/92 a total of 28 patients suffering from peripheral (n = 20) or centrally (n = 8) located soft tissue sarcomas had been were treated. Thirteen patients revealed a primary and 15 patients a recurrent tumor. Tumor resection with negative margins was performed in 20 patients, positive margins remained in 5 patients, and gross macroscopic residual disease in 3 patients. Combined intraoperative and external beam radiotherapy was applied in 22 patients, using IORT doses of 10-20 Gy and an external beam dose of 26-50 Gy. Three patients were irradiated intraoperatively twice with a time interval of 24 h. After a median follow-up of 9.9 months, 20 patients are disease free. Two patients died 4 and 5 months after the end of therapy with rapidly progressive distant metastases. An infield failure within the external beam target volume was seen in 1 patient and local failure at the field margin of the external field in 3 patients. So far, there have been no IORT infield failures. Follow-up is performed with magnetic resonance imaging. In 3 patients a second operation was necessary because of a severe wound infection, including one patient suffering from osteomyelitis of a neighboring bone. Mild sensory neuropathy occurred in 1 patient 7 months after treatment. Overall only mild and reversible postoperative and posttherapeutic complications were seen.(ABSTRACT TRUNCATED AT 250 WORDS)