Cloning, purification and characterization of non-human primate 12/15-lipoxygenases.

The enzyme 15-lipoxygenase-1 (15-LO-1) possesses mainly 15-LO activity and has so far only been described in human cells and rabbit reticulocytes. The animal ortholog, except rabbit reticulocytes, is an enzyme with predominantly a 12-lipoxygenase activity, commonly referred to as 12/15-LO. We describe herein the characterization of the 12/15-LOs in Macaca mulatta (rhesus monkey) and in Pongo pygmaeus (orang-utan). The rhesus and the orang-utan enzymes have mainly 12-lipoxygenase and 15-lipoxygenase activity, respectively, and they display 94% and 98% identity to the human 15-LO-1 protein. The rhesus enzyme was functionally different from the human enzyme with respect to substrate utilization in that anandamide was used differently and that the rhesus enzymes positional specificity could be affected by the substrate concentration. Furthermore, genomic data indicate that chimpanzees express an enzyme with mainly 15-lipoxygenase activity whereas marmosets express an enzyme with mainly 12-LO activity. Taken together, the switch during evolution from a 12-lipoxygenating enzyme in lower primates to a 15-lipoxygenating enzyme in higher primates and man might be of importance for the biological function of this enzyme.

Improved understanding of the effect of food on drug absorption and bioavailability for lipophilic compounds using an intestinal pig perfusion model.

The purpose of this study was to investigate the relative importance of mechanisms behind the effect of food on the intestinal absorption and bioavailability for low solubility compounds by applying a porcine single-pass perfusion model. Nanoparticle suspensions of the model compounds, danazol and cyclosporine were perfused through the jejunum in isotonic fluid alone (control) and isotonic fluid with a P-glycoprotein (P-gp) inhibitor (verapamil) or dietary and endogenous lipids added. The drugs were also administered as saturated solutions in the isotonic fluid containing lipids. Administration of cyclosporine together with verapamil increased the absorption compared to the control (1.6 times) suggesting an effect on jejunal permeability. However, addition of dietary lipids to the media led to a 50% reduction in the absorption of cyclosporine indicating lack of major effects by P-gp inhibition by lipids in vivo. The absorption of danazol was increased (2.6 times) when administered as a nanosuspension in lipid containing media compared to the control, but decreased (60%) when administered as a solution in the same media. This shows how important dissolution of the drug nanoparticles is in drug absorption. The difference in the effect of lipids in the absorption of cyclosporine and danazol when administered as nanosuspensions may be due to different distribution to the colloidal structures present in the media, thereby rendering the drugs' different diffusion rates in the perfused segment. In conclusion, solubilisation seems to be a more important factor than P-gp inhibition as an explanation for the food-drug interaction observed for several low solubility drugs. In addition, the partition into different colloidal structures seems to play a major role in the dissolution and absorption of poorly soluble drugs.

Rapid modulation of Ca2+ uptake in human jejunal enterocytes.

The active metabolite of D vitamin, 1,25(OH)2D3, has been suggested to promote acute uptake of calcium through the intestinal lining in cell lines and murine models. In this study, the effects of D vitamin on the cytoplasmic Ca2+ of single human jejunal enterocytes, obtained with LOC-I-GUT technique, was analyzed in vivo in a fluorometric system using fura-2 as the Ca2+-sensing probe. Vitamin-promoted acute Ca2+ influx exhibited dual kinetics, indicating initial release from intracellular Ca2+ pools and fast entry from the extracellular space. Furthermore, providing a chemical clamp of membrane potential close to 0 mV did not activate voltage-sensitive calcium channels in the cellular membrane, neither was the hormone-induced Ca2+ influx affected by verapamil. This advocates that voltage-operated channels like L-type Ca2+ channels do not participate in the process of Ca2+ uptake. In fact, the existence of calcium-release-activated-calcium channels (I(CRAC)) was implied by the findings that irreversible depletion of intracellular Ca2+ stores by thapsigargin promoted Ca2+ entry. In the thapsigargin-treated enterocytes, D vitamin lost its ability to promote calcium entry indicating an important role for intracellular store-operated Ca2+ stores in the acute effects of 1,25(OH)2D3.

Purification, characterization, and cDNA sequencing of cytosolic phospholipase A(2) from equine neutrophils.

It has been demonstrated that equine neutrophils, but not eosinophils, require exogenous arachidonic acid for calcium ionophore A23187-induced leukotriene synthesis. Because cytosolic phospholipase A(2) (cPLA(2)) plays an essential role in leukotriene formation in leukocytes, we investigated the presence of a functional cPLA(2) in equine neutrophils. To determine whether cPLA(2) from neutrophils was catalytically active, we purified the enzyme >6,500 fold with 3% recovery from equine neutrophils. The full-length cDNA sequence encoded a 749-amino acid protein. The deduced amino acid sequence demonstrated 95% identity with human and mouse cPLA(2), as well as 83 and 73% identity with chicken and zebra fish cPLA(2) protein, respectively. The equine cPLA(2) possessed some properties that distinguished the equine enzyme from the human enzyme. First, the enzyme activity of the equine cPLA(2) was differently influenced by cations as compared with the human cPLA(2). Second, the equine neutrophil cPLA(2) migrated as an approximately 105-kDa protein, in comparison with human cPLA(2) which migrated as a 110-kDa protein. A difference between equine neutrophils and eosinophils in the degree of phosphorylation of the cPLA(2) protein was observed. Thus, the cPLA(2) protein from eosinophils was constitutively phosphorylated, while the cPLA(2) protein from neutrophils was unphosphorylated. In summary, these results demonstrate that equine neutrophils indeed express an active cPLA(2) protein but that there is a difference in the degree of phosphorylation of the cPLA(2) protein between equine neutrophils and eosinophils. This difference might explain the difference between the two cell types in the capacity to produce leukotrienes from endogenous substrate.

[Laparoscopic gastric banding]. / Laparoskopisches gastric banding.

The introduction of laparoscopic gastric banding appears to have revolutionized bariatric surgery. This review presents this new method in terms of indications, operative technique and preliminary results according to our own experience as well as reports in the literature. It seems that a long-term weight reduction of more than 50% excess weight can be achieved with a low morbidity rate. In the near future laparoscopic gastric banding can possibly become the procedure of choice for the treatment of morbid obesity.

The Swedish Adjustable Gastric Band: laparoscopic technique and preliminary results.

BACKGROUND: The laparoscopic technique for the Swedish Adjustable Gastric Band (SAGB) has been developed based on the previously established open technique. METHODS: From March 1996-June 1997, laparoscopic SAGB was attempted in 85 consecutive obese patients (77 women and 8 men). The average preoperative BMI was 44 (34-59). RESULTS: All operations except one were completed by laparoscopy. One patient had to be converted because of unfavorable anatomic conditions. The average operating time was 40 minutes. There were no immediate perioperative complications. All patients were followed for 1 year. During this period 2 patients developed esophagitis and 3 patients experienced repeated vomiting. There were no other complications. At 1 year follow-up the average BMI was 33 (21-46). The excess weight loss was 54% (17-100%). CONCLUSION: Early results are encouraging. No significant complications related to the technique were registered. One-year weight loss was equal to what was achieved by open surgery. Laparoscopic SAGB will be established as an attractive alternative for surgical treatment of morbid obesity.

Genomic characterization of the human and mouse protein tyrosine phosphatase-1B genes.

PTP-1B is a ubiquitously expressed intracellular protein tyrosine phosphatase (PTP) that has been implicated in the negative regulation of insulin signaling. Mice deficient in PTP-1B were found to have an enhanced insulin sensitivity and a resistance to diet-induced obesity. Interestingly, the human PTP-1B gene maps to chromosome 20q13.1 in a region that has been associated with diabetes and obesity. Although there has been a partial characterization of the 3' end of the human PTP-1B gene, the complete gene organization has not been described. In order to further characterize the PTP-1B gene, we have cloned and determined the genomic organization for both the human and mouse PTP-1B genes including the promoter. The human gene spans >74 kb and features a large first intron of >54 kb; the mouse gene likewise contains a large first intron, although the exact size has not been determined. The organization of the human and mouse PTP-1B genes is identical except for an additional exon at the 3' end of the human that is absent in the mouse. The mouse PTP-1B gene maps to the distal arm of mouse chromosome 2 in the region H2-H3. This region is associated with a mouse obesity quantitative trait locus (QTL) and is syntenic with human chromosome 20. The promoter region of both the human and mouse genes contain no TATA box but multiple GC-rich sequences that contain a number of consensus SP-1 binding sites. The basal activity of the human PTP-1B promoter was characterized in Hep G2 cells using up to 8 kb of 5' flanking sequence. A 432 bp promoter construct immediately upstream of the ATG was able to confer maximal promoter activity. Within this sequence, there are at least three GC-rich sequences and one CCAAT box, and deletion of any of these elements results in decreased promoter activity. In addition, the promoter in a number of mouse strains contains, 3.5 kb upstream of the start codon, an insertion of an intracisternal a particle (IAP) element that possibly could alter the expression of PTP-1B mRNA in these strains.

The human calcium-independent phospholipase A2 gene multiple enzymes with distinct properties from a single gene.

Recently, we reported the human 88-kDa calcium-independent phospholipase A2 (iPLA2) cDNA sequence, as well as extensive alternative splicing of the iPLA2 mRNA. In this report we identified the gene coding for iPLA2, which was localized on chromosome 22q13.1. The gene consists of at least 17 exons spanning > 69 kb. Based on the iPLA2 gene organization the splice variants can be explained. The putative promotor for the iPLA2 gene lacks a TATA-box and contains a CpG island as well as several potential Sp-1-binding sites. Furthermore, the 5'-flanking region also contains one medium reiteration frequency repeat (MER53) and an Alu repetitive sequence. Northern blot analysis of iPLA2 mRNA in various human tissues demonstrated tissue-specific expression of four distinct iPLA2 transcripts. The native human 3.2-kb iPLA2 transcript was predominantly expressed in heart, brain, skeletal muscle, prostate, testis, thyroid and spinal cord, and to a lesser extent in peripheral blood leucocytes, stomach, trachea and bone marrow. Studies on the subcellular localization of the native iPLA2 protein were performed in COS-7 cells overexpressing this enzyme. The cytosolic fraction of untransfected and cells overexpressing iPLA2 contained equal amounts of calcium-independent PLA2 activity. However, the membrane fraction displayed a 5.5-fold increased activity in iPLA2 overexpressing cells. This increased calcium-independent PLA2 activity correlated with the presence of iPLA2 immunoreactive protein in the membrane fraction, indicating that this form of iPLA2 protein was membrane associated. Studies of iPLA2 in rat vascular smooth muscle cells verified the membrane association of this form of iPLA2. The major difference between this form of iPLA2 enzyme and the soluble forms of iPLA2 studied previously is the presence of 54 additional amino acid residues derived from exon 9. We suggest that the addition of these 54 amino acids leads to a membrane-associated protein. In summary, these results demonstrate that alternative splicing of the human iPLA2 transcript generates multiple iPLA2 isoforms with distinct tissue distribution and cellular localization.

Complications following Swedish adjustable gastric banding: a long-term follow-up.

BACKGROUND: The Swedish adjustable gastric band (SAGB) has been in use since 1985. The aim of this study was to analyze short and long-term complications linked to the SAGB. MATERIALS AND METHODS: Between August 1990 and December 1996, we operated on a series of 326 patients (78 men and 248 women) at the Huddinge University Hospital and the Norra Alvsborg County Hospital. The mean age of patients at surgery was 40 years (range 19-62). The mean preoperative weight was 125 kg (range 81-181). The mean excess weight was 80%. RESULTS: The mean time of follow-up was 28 months (range 6-76). Complications requiring reoperation included two (0.6%) band dislocations, six (1.8%) band leakages, and 16 (4.6%) band migrations-erosions. The most common reason for abdominal reoperation, band migration, was attributed to overfilling of the band system. In the patients in whom migration occurred, the bands had been filled with a mean volume of 12.6 ml fluid. In the remaining patients, the mean volume was 8.7 ml. The most common complication not requiring reoperation was reflux disease (4.7%). In cases with a small pouch, this complication did not seem to be a serious problem. The mean excess weight loss in the 296 patients without complications was 68%. CONCLUSION: The overall long-term complication rate following SAGB is reasonable. With improved operating technique and closer follow-up, it should be possible to reduce the complication rate further. Reoperation because of band migration appears to be related to overfilling of the system and should therefore be avoidable in most cases.

On the expression of cytosolic calcium-independent phospholipase A2 (88kDa) in immature and mature myeloid cells and its role in leukotriene synthesis in human granulocytes.

The human calcium-independent phospholipase A2 (iPLA2; 88 kDa) has recently been cloned (Larsson, P.K.A., Claesson, H.-E. and Kennedy, B.P. (1998) J. Biol. Chem. 272, 207-214). Here we demonstrate the expression of the human iPLA2 mRNA and its splice variants in blood progenitor cells, immature leukemic cells and mature granulocytes. Chromatographical resolvable iPLA2 activity was found in the cytosolic fraction of granulocytes and the activity was inhibited by the iPLA2 inhibitor bromoenol lactone. This drug also inhibited leukotriene synthesis in human granulocytes, induced by low concentration of calcium ionophore A23187 (0.10-0.15 microM) or opsonized zymosan. These results suggest that iPLA2 is involved in the regulation of the pool of arachidonic acid destined for leukotriene synthesis in human granulocytes.

The Swedish Adjustable Gastric Banding (SAGB) for morbid obesity: 9 year experience and a 4-year follow-up of patients operated with a new adjustable band.

BACKGROUND: We have developed an adjustable gastric band in which the stoma diameter can be adjusted from the outside. A standardized technique was employed and the application of our band in terms of weight loss and complication rate was evaluated METHODS: Between August 1990 and November 1991, 50 patients (15 men and 35 women) were operated on by laparotomy. Their mean age at surgery was 41 (19-60) years. Mean preoperative weight was 134 (106-181) kg and the mean BMI was 46 kg/m2 (range 33-59 kg/m2). RESULTS: No patient was lost to follow-up. Four were excluded from the study (brain tumor, pregnancy and two reoperations). The remaining 46 were followed for at least 4 years. At follow-up, mean weight was 80 kg and mean BMI was 27.5 kg/m2. The patients had lost a mean of 54 kg. Two patients (4%) had abdominal reoperation because of technical problems. There was one incisional hernia and one minor wound infection, but no other significant complications. CONCLUSION: This relatively simple method appears to be at least as good as the other operations, and weight loss can be adjusted to patient comfort. Currently, the procedure is being performed laparoscopically.

Hepatic cholesterol metabolism in human obesity.

Hepatic cholesterol metabolism was studied in operative liver biopsies from 17 morbidly obese subjects and compared with that in samples from 15 nonobese controls. The aim was to understand the mechanisms causing the hypersecretion of cholesterol into bile. The content of cholesteryl esters was increased threefold in the liver of obese subjects compared with that of the controls (P < .0001). The activity and the messenger RNA (mRNA) level of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, the rate limiting enzyme for cholesterol synthesis, were higher in the obese subjects compared with the nonobese subjects (75% and 140%, respectively; P < .01). In the obese subjects, the activity and mRNA level of cholesterol 7alpha-hydroxylase, which regulates the catabolism of cholesterol to bile acids, were also increased by 140% (P < .05) and 180% (P = .06), respectively, as compared with the controls. There was a significant correlation between the activities and the mRNA levels of cholesterol 7alpha-hydroxylase among the obese subjects (r = +0.65, P < .01). The activities of acyl-coenzyme A:cholesterol acyltransferase (ACAT), which governs cholesteryl ester formation, in obese and nonobese patients were 12.5 +/- 1.7 and 8.1 +/- 1.2 pmol/min/mg protein, respectively (P < .05), and the low-density lipoprotein (LDL) receptor mRNA levels were 5.3 +/- 0.7 and 4.5 +/- 0.9 molecules of mRNA/microg of RNA, respectively. We conclude that the activities of three key enzymes in hepatic cholesterol metabolism were increased in morbidly obese subjects compared with nonobese controls, as were mRNA levels of HMG CoA reductase and cholesterol 7alpha-hydroxylase. The mRNA level of the LDL receptor in the obese subjects was not significantly changed. The hypersecretion of cholesterol occurring in obesity is neither due to a reduced conversion of cholesterol to bile acids nor to a decreased esterification of hepatic cholesterol but may be due to an increased synthesis of cholesterol.

Validation of Pouch Size Measurement Following the Swedish Adjustable Gastric Banding Using Endoscopy, MRI and Barium Swallow.

BACKGROUND: Pouch volume appears to be of major importance for subsequent weight loss following any gastric restriction type of surgery for morbid obesity. In order to be able to evaluate pouch volume following Swedish Adjustable Gastric Banding (SAGB), an endoscopic pouch volume classification system was designed in which pouch volume is classified in five categories. The aim of this study was to validate the endoscopic classification system using MRI and barium swallow as reference methods for pouch volume measurement. METHODS: Twenty patients (13 women and seven men) were operated for obesity with SAGB. They were investigated a mean of 3 years (6 weeks-5.5 years) after surgery and had at that time lost a mean of 60 (12-112) kg. During the same afternoon they sequentially underwent endoscopy, MRI and barium swallow with an empty stomach. RESULTS: The mean pouch volume measured with MRI was 70 ml (0-1 80 ml) and with barium swallow was 72 ml (0-1 95 ml). In 17/20 patients the volume as measured by MRI and barium swallow was in the same volume category as with endoscopy. The correlation measured according to Pearson was significant between endoscopy on one hand and MRV barium swallow both independently and together (p < 0.001). CONCLUSION: Based on these results we are confident in using our endoscopic classification system for postoperative follow-up of pouch volume.

Pouch Volume, Stoma Diameter and Weight Loss in Swedish Adjustable Gastric Banding (SAGB).

BACKGROUND: Weight loss appears to be inversely related to pouch volume following gastric restriction procedures for morbid obesity. The aim of this study was to investigate the changes in pouch volume with time and the relationship between pouch volume and stoma diameter and subsequent weight loss following the Swedish Adjustable Gastric Banding (SAGB). METHODS: During 1990 50 patients were operated upon. Their mean BMI at surgery was 46 and at 2 years 28. We followed these patients with endoscopy at 6 weeks and 3, 18 and 24 months after surgery. During endoscopy pouch volume was estimated according to a standardized classification system and measured stoma diameter using balloon catheters. RESULTS: The results indicate that the pouch dilates during the first few months after surgery but that the size thereafter is fairly stable. There is also a relationship between pouch volume and subsequent weight loss. Pouch volume seems to be the primary determinator for weight loss. CONCLUSION: The smaller the pouch the greater the weight loss. Reduction of the stoma diameter is a good instrument for regulating the degree and speed of weight loss in patients with small pouches, but much less powerful in patients with large pouches.

Vertical Banded Gastroplasty with Adjustable Silicone Band: Preliminary Experience.

A series of ten patients operated on with vertical banded gastroplasty (VBG) with an adjustable silicone band at the outlet is presented. The loss of body weight and complication rate is evaluated. Preoperative mean excess overweight of the patients was 94% and mean BMI was 42.6. The loss of body weight at one year's follow-up was 38 kg or 59% of excess weight. Complications were one case of infection at the subcutaneous injection port and one case of a nonfatal pulmonary embolus. The results so far are thus comparable with VBG with a conventional fixed band, but the adjustable band actually simplifies the operative procedure since no exact calibration of the collar size is necessary at the time of surgery and should diminish the need for reoperations due to misalignment of collar size. The possibility of better weight control in the long-term perspective remains to be proven.

Gastric Banding for Morbid Obesity: Initial Experience with a New Adjustable Band.

A new adjustable gastric band was developed, consisting of a silicone balloon connected to a subcutaneous port In a closed system. The stoma diameter can be regulated within an extensive range (0-40 mm). The diameter is adjusted individually for each patient and weight loss can therefore be controlled and optimized. We evaluated the application of this new gastric banding procedure in terms of technical feasibility, complication rate and weight loss, and also the relationship between weight loss and pouch volume. Between January 1987 and April 1990 two preliminary studies of 18 and 24 patients respectively were carried out. In the first group there were technical problems resulting in insufficient weight loss. We therefore changed the procedure. In the second group the system thereafter worked as expected. In the second group mean preoperative weight was 132 kg, mean excess weight 60 kg, and mean BMI 45. The mean follow-up was in 21 months. At follow-up mean weight was 91 kg, mean weight loss 41 kg, and mean BMI 31. The mean postoperative stay was 6.0 days. Pouch volume and stoma diameter were followed by regular ondoscopy. There was a distinct relationship between pouch volume and weight loss-the smaller the volume the greater the weight loss.

A Gastric Band with Adjustable Inner Diameter for Obesity Surgery: Preliminary Studies.

Gastric banding for morbid obesity is theoretically an attractive method, since it is easily reversible, does not require opening of the stomach or intestines, and is associated with a very low surgical risk. The disadvantage is the high rate of reoperation because of the difficulty to obtain an optimal stoma diameter. This led us to develop a new gastric balloon band in which it is possible to regulate the inner diameter between 0 and 40 mm. In this study of the technical properties, we have investigated the inner pressure of the band during filling, the breaking point of the balloon when overfilling, the strength of the band and finally the degree of diffusion through the system. We found that there is no pressure with filling up to 10 ml. The band is thus a low pressure system. It can be filled with five times its normal volume before breaking. A pressure of 300 mmHg can be applied inside the system without breaking it. Finally, when the system was filled with soya oil, there was no detectable diffusion through the system in a one-year trial. These findings are consistent with our preset specifications. Clinical trials have therefore been started.

Transferability of a computer system for medical history taking and decision support in dyspepsia. A comparison of indicants for peptic ulcer disease.

The transferability of a British data base for differential diagnosis of dyspepsia using data obtained by computer interrogation was tested in 467 Swedish patients. The diagnostic value for peptic ulcer disease of symptoms such as frequent night pain relieved by food or antacids, smoking, family history of ulcer, food relief pain, male sex, and episodic pain was shown to be reproducible. However, for a number of symptoms their value for the diagnosis of peptic ulcer disease could not be reproduced in Swedish patients. The combined value of indicants was tested using a computer based algorithm for calculating diagnostic probabilities. The performance of this algorithm was poor when British data were applied to Swedish patients but reclassification of the Swedish patients on their own data base showed promising results. Crean and colleagues in Glasgow have developed a computer system for automated interrogation of patients with dyspepsia. The system utilises a large number of questions to obtain information regarding a maximum of 160 diagnostic indicants. The symptoms elicited from a patient can be compared with those of a large number of previously examined patients and the probabilities of ten different diagnoses can be calculated. The calculation of diagnostic probabilities is based on scores reflecting the diagnostic value of different symptoms in different diseases. After careful translation of questions the system has been transferred for use in Sweden. The present report is based on data from patients seen during the first two years with the system at a Swedish hospital.(ABSTRACT TRUNCATED AT 250 WORDS)

Perforating appendicitis. A nine-year survey of treatment and results.

This is a clinical study of 193 adult cases of perforating appendicitis during a nine-year period of treatment. There were 77 females (39.9%) and 116 males (60.1%) with ages of 15 years and above. Throughout the study period, the yearly perforation frequency ranged from 10.0 to 23.1% (mean 16.7%) of all cases with proven appendicitis. Perforation was equally common in both sexes, females: 15.5%, males: 17.6% of the appendicitis cases. In elderly patients (greater than or equal to 60 years of age) perforation was much more common; 37 out of 77 cases with proven appendicitis (48.1%). Compared to different ages (patients under and above 60 years of age), clinical and laboratory findings were found equal. Duration of symptoms showed no differences for younger and elderly patients. Preoperative delay in the hospital of at least 6 hours occurred in 39.9% of all patients, and was found equally common in patients younger than 60 years (39.1%) and in those from 60 years and older (43.2%). There were no fatalities in the study group. Postoperative septical complications predominated over non-septical (32.6 versus 9.3%), and were equally common in both sexes and in patients of different ages. The frequency of occurrence of septical complications was not significantly influenced by the duration of symptoms or by the duration of operation, furthermore, septical complications were equally frequent throughout the study period, although the use of antibiotics changed towards drugs more potent to cover anaerobic bacteria (28.1% infections for the period 1972-1976 versus 29.8% for the period 1977-1981).(ABSTRACT TRUNCATED AT 250 WORDS)