Suppression of adverse angiogenesis in an albumin-based hydrogel for articular cartilage and intervertebral disc regeneration.

An injectable polyethylene glycol-crosslinked albumin gel (AG) supplemented with hyaluronic acid as a matrix for autologous chondrocyte implantation was evaluated with regard to its impact on angiogenesis. Healthy articular cartilage and intervertebral discs (IVD) are devoid of blood vessels, whereas pathological blood vessel formation augments degeneration of both theses tissues. In contrast to human endothelial cells, primary human articular chondrocytes encapsulated in the AG retained their viability. Endothelial cells did not adhere to the gel surface to a significant extent nor did they proliferate in vitro. The AG did not release any diffusible toxic components. Contrary to Matrigel employed as positive control, the AG prevented endothelial chemoinvasion in Transwell filter assays even in the presence of a chemotactic gradient of vascular endothelial growth factor. In ovo, the AG exhibited a barrier function for blood vessels of the chick chorioallantoic membrane. Subcutaneous implantation of human IVD chondrocytes enclosed in the albumin gel into immunodeficient mice revealed a complete lack of angiogenesis inside the gel after two weeks. At the same time, the IVD chondrocytes within the gel remained vital and displayed a characteristic gene expression pattern as judged from aggrecan, collagen type I and type II mRNA levels. In summary, aiming at articular cartilage and IVD regeneration the albumin gel promises to be a beneficial implant matrix for chondrocytes simultaneously exhibiting non-permissive properties for adverse endothelial cells.

[Endoluminal therapy of the trachea and bronchus]. / Die endoluminale Therapie in Trachea und Bronchus.

The application of endoscopic techniques is common in the treatment of tracheal and bronchial diseases today. Bronchoscopic interventions are used in both elective and emergency situations. Laser therapy for malignant tumors is purely palliative in most cases and should only be performed in nonsurgical patients. However, 30% of lung cancers cause obstruction in the trachea and main bronchi. Benign tumors and tracheal stenoses could require laser recanalization or the implantation of stents, if surgery will be the second step or will not be possible. In patients with foreign body aspiration, massive hemoptysis, or severe obstruction of the trachea, emergency bronchoscopy is necessary. A more recent type of bronchoscopic intervention is the treatment of bronchial stump or anastomosis insufficiency as well as minimal iatrogenic injuries using spongiotic fillings or stent implantation. The use of therapeutic bronchoscopy requires great experience in rigid and flexible bronchoscopy, the possibility of high-frequency jet ventilation as well as laser and argon application, and the possibility to implant different types of stents. More advanced bronchoscopic interventions should only be done if a department of thoracic surgery exists, in view of the potential need to control complications or perform further treatment. Especially the bronchoscopic treatment of tracheal stenosis should be performed by the thoracic surgeon himself or in close contact with a thoracic surgeon who is experienced in tracheal resections.

Reduced blood loss by aprotinin in thoracic surgical operations associated with high risk of bleeding. A placebo-controlled, randomized phase IV study.

OBJECTIVE: Although the blood-saving effect of aprotinin has been well documented in cardiac surgery and lung transplantation, its use in lung surgery has received less attention. We present our experience with the intraoperative application of aprotinin in lung resections with a predicted high risk of bleeding. METHODS: Thirty-eight patients undergoing major thoracic surgical procedures were randomized into treatment and placebo groups. The treatment group (n=18) received a bolus of 2 x 10(6) kallikrein inhibitor units (KIU) of aprotinin followed by 5 x 10(5) KIU/h during surgery. The placebo group (n=20) received an isotonic saline infusion instead. RESULTS: There was no significant difference between the groups concerning diagnosis, co-morbidity, age, sex, height, and weight. The mean intraoperative blood loss in the treatment group was significantly reduced (342 vs. 808 ml, P<0024), postoperative blood loss was also reduced (623 vs. 1282 ml, P<0.0007) and the need for blood transfusion was less (14 vs. 60, n.s.). No severe side effects of aprotinin were registered. Re-thoracotomy was necessary in two patients of the placebo group because of postoperative bleeding. CONCLUSION: Aprotinin reduces the perioperative blood loss and the need for blood transfusion in thoracic surgical procedures in patients with an increased risk of bleeding.

[CO2 stores in laparoscopic cholecystectomy with CO2 pneumoperitoneum]. / Zur Frage der CO2-Speicherung bei laparoskopischer Cholezystektomie mit CO2-Pneumoperitoneum.

METHODS: Two groups of 22 patients each were studied in a prospective, randomised fashion during laparoscopic cholecystectomy (LCh) and CO2 pneumoperitoneum (PP) with regard to end-tidal and arterial PCO2 and pulmonary elimination of CO2 (ECO2, Servoventilator with integrated CO2-analyser 930, Siemens). In group 1 minute ventilation was kept constant, resulting in moderate hypercapnia during PP. paCO2 increased by about 10 mmHg during up to 50 min PP. In group 2 paCO2 was kept constant by a stepwise increase in minute ventilation (Fig. 1, Table 2). RESULTS: Compared to values just before PP, ECO2 increased in group 1 rather rapidly up to 20 min of PP and more slowly thereafter, reaching a mean value 35% above control at 45 min PP. In group 2 ECO2 was significantly higher than in group 1 between 15 and 35 min PP. At 45 min PP, however, ECO2 was identical in both observation groups (Fig. 2). CONCLUSIONS: Assuming a stable metabolic CO2 production rate during the observation period and no differences in CO2 absorption from the PP between the two study groups, differences in ECO2 between groups would be a measure of CO2 stored in group 1 patients during the increase in paCO2 with PP (Fig. 3, Table 3). CO2 storage rapidly increased between 0 and 15 min PP, more or less reached a plateau between 15 and 35 min PP, and ceased at 45 min PP. Storing capacity for CO2 during the first 45 min PP amounted to a mean value of 1.20 ml CO2/kg body weight and mmHg paCO2, which agrees favourably with data from the literature and a computer model from Fahri and Rahn published in 1960 (Fig. 4, Table 4). If during LCh with CO2-PP patients are ventilated with a constant minute ventilation, a moderate increase in paCO2 of about 10 mm Hg can be expected. In this case, during the first 45 min PP a 70-kg patient will retain about 1000 ml CO2 in blood and tissues, which must be eliminated after cessation of PP. If the paCO2 is to be held constant during PP, minute ventilation has to be progressively increased by about 40%.

The gene for a major exopolyphosphatase of Saccharomyces cerevisiae.

The gene encoding a major exopolyphosphatase (scPPX1) in Saccharomyces cerevisiae (H. Wurst and A. Kornberg, J. Biol. Chem. 269:10996-11001, 1994) has been isolated from a genomic library. The gene, located at 57 kbp from the end of the right arm of chromosome VIII, encodes a protein of 396 amino acids. Overexpression in Escherichia coli allowed the ready purification of a recombinant form of the enzyme. Disruption of the gene did not affect the growth rate of S. cerevisiae. Lysates from the mutants displayed considerably lower exopolyphosphatase activity than the wild type. The enzyme is located in the cytosol, whereas the vast accumulation of polyphosphate (polyP) of the yeast is in the vacuole. Disruption of PPX1 in strains with and without deficiencies in vacuolar proteases allowed the identification of exopolyphosphatase activity in the vacuole. This residual activity was strongly reduced in the absence of vacuolar proteases, indicating a dependence on proteolytic activation. A 50-fold-lower protease-independent activity could be distinguished from this protease-dependent activity by different patterns of expression during growth and activation by arginine. With regard to the levels of polyP in various mutants, those deficient in vacuolar ATPase retain less than 1% of the cellular polyP, a loss that is not offset by additional mutations that eliminate the cytosolic exopolyphosphatase and the vacuolar polyphosphatases dependent on vacuolar protease processing.

[CO2--emphysema in laparoscopic surgery. Changes in pulmonary CO2-elimination]. / CO2-Emphysem bei laparoskopischer Chirurgie. Veränderungen der pulmonalen CO2-Elimination.

We report on two patients with subcutaneous carbon dioxide (CO2) emphysema that developed during laparoscopic surgery with CO2 pneumoperitoneum (PP), in whom pulmonary elimination of CO2 (ECO2, Servo ventilator with integrated CO2 analyzer 930, Siemens) was continuously monitored. Patient 1 was a 61-year-old man with laparoscopic herniotomy. ECO2 immediately before PP was 120 ml/min x m2 and increased rapidly after 45 min PP to a maximum value of 340 ml/min x m2. At that time, minute ventilation had been increased from 7 to 11 l/min and PaCO2 had risen from 35 to 57 mm Hg. At the end of the procedure the patient showed excessive subcutaneous emphysema. Patient 2 was a 71-year-old woman in whom diagnostic laparoscopy was performed for staging of a pancreatic tumor. ECO2 immediately before PP was 140 ml/min x m2, increasing dramatically after 45 min PP to a maximum value of 529 ml/min x m2 (Fig. 1). At that time minute ventilation had been increased from 6.2 to 12.5 l/min and PaCO2 had risen from 40 to 77 mm Hg. PP was terminated and the patient was found to have extreme subcutaneous emphysema. She was mechanically ventilated for a further 40 min to normalize PaCO2 and ECO2. It seems reasonable to suppose that an increase in ECO2 by more than 100% of control during CO2-PP is an early sign of CO2 emphysema. In this situation hypercapnia is potentially life-threatening. Evidently, reabsorption of CO2 from loose connective tissue is far more rapid and effective than CO2 resorption from the peritoneal cavity.(ABSTRACT TRUNCATED AT 250 WORDS)

A soluble exopolyphosphatase of Saccharomyces cerevisiae. Purification and characterization.

A soluble polyphosphatase of Saccharomyces cerevisiae, purified to apparent homogeneity, is monomeric with a molecular mass of 40 kDa. It acts as an exoenzyme in a processive mode releasing orthophosphate residues from long polyphosphate chains until pyrophosphate is reached. Polyphosphates of all the lengths examined are used as substrates with a preference for those of about 250 residues. These are degraded with a kcat/Km near the limit for diffusion-controlled reactions. At 37 degrees C, the enzyme releases about 500 phosphate residues/s. It does not act on pyrophosphate, ATP, or the cyclic form of tripolyphosphate. For optimal activity the enzyme requires magnesium, manganese, or cobalt.

[Pulmonary CO2 elimination in laparoscopic cholecystectomy. A clinical study]. / Pulmonale CO2-Elimination bei laparoskopischer Cholezystektomie. Eine klinische Untersuchung.

METHODS: We measured pulmonary elimination of carbon dioxide (VCO2), end-tidal and arterial CO2 tensions (PETCO2, PaCO2), deadspace ventilation (VD/VT), and arterial oxygen tension (PaO2) using a Siemens 930 CO2 analyzer incorporated into a servoventilator and arterial blood gas analyses, respectively, in 31 patients undergoing laparoscopic cholecystectomy with a median duration of pneumoperitoneum (PP) of 60 min. RESULTS: During the first 30 min of PP VCO2 increased significantly by a mean of 30% (Fig. 1). At the same time, with constant minute ventilation PETCO2 und PaCO2 increased by about 8 mm Hg each (Fig. 3, Table 1). In a subgroup of 10 patients who could be observed for up to 75 min of PP, we found a stepwise increase in minute ventilation with no further increase in PETCO2 and PaCO2 after 30 min PP, but a slowly rising VCO2 (Fig. 2). Arterial-to-end-tidal CO2 tension difference (Pa-PETCO2) remained constant at about 4 mm Hg with institution and during the course of PP (Fig. 4), as did VD/VT at a median value of 0.38-0.40 (Fig. 5). PaO2 (FIO2 = 0.5) did not change significantly with PP (Table 1). With desufflation we found a short-term increase in VCO2 (Table 2). CONCLUSIONS: During PP, CO2 is reabsorbed from the peritoneal cavity. During the initial unstable phase with rising PaCO2, reabsorption of CO2 is the sum of increased pulmonary elimination of CO2 above baseline and uptake of CO2 into gas stores of the body. We estimated CO2 reabsorption to be on the order of 70 ml/min during the first 30 min of PP. During the later, stable phase, reabsorption of CO2 equals increased pulmonary elimination of CO2 above baseline and was estimated to be in the order of 90 ml/min in 10 patients with 30-75 min of PP (hatched area in Fig. 2). PET-CO2 corresponded well with PaCO2 in these patients. VD/VT and arterial oxygenation did not change significantly with institution or during the course of PP. Monitoring VCO2 probably is a useful aid in the early detection of CO2 emphysema (Fig. 6).

Genomic sequencing by ligation mediated polymerase chain reaction using direct blotting and non-radioactive detection.

Genomic sequencing has become an important tool for analyzing uncloned cellular DNA with regard to the methylation status of cytidines as well as to DNA-protein interactions within cells. The hybridization step of the genomic sequencing procedure requires a very high sensitivity, rendering the method fairly difficult. Using a modified ligation mediated polymerase chain reaction procedure (LMPCR) and a sensitive non-radioactive detection method, we have developed a procedure avoiding the high amounts of radioactivity formerly needed for detection of chemically cleaved genomic DNA. The detection limit of our method of genomic sequencing is less than 1 microgram mammalian DNA, which is much better than the detection limit of the original genomic sequencing method and comparable with the detection limit of radioactive detection after the LMPCR procedure. In addition to the advantages of the non-radioactive detection technique we simplified the blotting step of the genomic sequencing procedure by using the direct blotting electrophoresis method. The method was applied to a region 5' to the human c-myc promoter of HeLa cells and was able to verify the sequence obtained by other authors and to specify the methylation status of five CpG-pairs within this sequence.

Mutant profiles of selectable genetic elements.

A method is presented that allows simultaneous analysis of the effects of all possible point mutations within a specific mutation window of at least 50 base pairs on a DNA fragment that codes for a selectable function. It relies on the detection of mismatched base pairs with hydroxylamine and osmium tetroxide. A mutant plasmid library of randomly distributed point mutations within the lacZ' gene of Escherichia coli was selected for functional alpha-complementation by growth on lactose. The DNA fragments of the selected and unselected library were each heat denatured and again renatured, thereby generating a randomly distributed set of all possible mismatches within the mutagenesis window. Cytidine-containing mismatches were then detected with hydroxylamine, and thymidine-containing mismatches were detected with osmium tetroxide. When this procedure was performed for both DNA strands, all mismatches could be detected. A comparison of the results of the unselected and selected library leads to an estimation of the effects of each detectable mutation on alpha-complementation in vivo. This method, called "mutant profiling," should be applicable to all selectable genetic elements.

DNA sequencing with direct blotting electrophoresis and colorimetric detection.

We describe optimized procedures for colorimetrically-detected DNA sequencing with direct blotting electrophoresis. One-step protocols for Sequenase and Klenow enzyme are given. The clapping technique has been adapted to allow convenient casting of very thin gels with an optimal lower gel (transfer) surface. This gives very sharp band patterns, enabling more than 350 bases from a single loading to be read with confidence. The crucial points for direct blotting electrophoresis are discussed. Background problems resulting from unspecific binding of streptavidin to the nylon membranes have been eliminated by the use of high concentrations of SDS in the incubation buffer; and using a single large glass tube for all incubation and washing steps is a very convenient and effective development protocol. Automation of the colorimetric development process is described.