Fat-induced membrane cholesterol accrual provokes cortical filamentous actin destabilisation and glucose transport dysfunction in skeletal muscle.

AIMS/HYPOTHESIS: Diminished cortical filamentous actin (F-actin) has been implicated in skeletal muscle insulin resistance, yet the mechanism(s) is unknown. Here we tested the hypothesis that changes in membrane cholesterol could be a causative factor, as organised F-actin structure emanates from cholesterol-enriched raft microdomains at the plasma membrane. METHODS: Skeletal muscle samples from high-fat-fed animals and insulin-sensitive and insulin-resistant human participants were evaluated. The study also used L6 myotubes to directly determine the impact of fatty acids (FAs) on membrane/cytoskeletal variables and insulin action. RESULTS: High-fat-fed insulin-resistant animals displayed elevated levels of membrane cholesterol and reduced F-actin structure compared with normal chow-fed animals. Moreover, human muscle biopsies revealed an inverse correlation between membrane cholesterol and whole-body glucose disposal. Palmitate-induced insulin-resistant myotubes displayed membrane cholesterol accrual and F-actin loss. Cholesterol lowering protected against the palmitate-induced defects, whereas characteristically measured defects in insulin signalling were not corrected. Conversely, cholesterol loading of L6 myotube membranes provoked a palmitate-like cytoskeletal/GLUT4 derangement. Mechanistically, we observed a palmitate-induced increase in O-linked glycosylation, an end-product of the hexosamine biosynthesis pathway (HBP). Consistent with HBP activity affecting the transcription of various genes, we observed an increase in Hmgcr, a gene that encodes 3-hydroxy-3-methyl-glutaryl coenzyme A reductase, the rate-limiting enzyme in cholesterol synthesis. In line with increased HBP activity transcriptionally provoking a membrane cholesterol-based insulin-resistant state, HBP inhibition attenuated Hmgcr expression and prevented membrane cholesterol accrual, F-actin loss and GLUT4/glucose transport dysfunction. CONCLUSIONS/INTERPRETATION: Our results suggest a novel cholesterolgenic-based mechanism of FA-induced membrane/cytoskeletal disorder and insulin resistance.

Cyclooxygenase 1 is required for pH control at the mouse gastric surface.

BACKGROUND: Endogenous cyclooxygenase (COX) activity is required to maintain a relatively alkaline surface pH at the gastric luminal surface. AIMS: The purpose of this study was to determine which COX isoform, COX-1 or COX-2, is responsible for regulating the protective surface pH gradient and to test if COX inhibitors also had non-COX mediated effects in vivo. METHODS: Immunofluorescence and western blot analysis showed constitutive expression of both COX isoforms in the normal mouse stomach. We used in vivo confocal microscopy to measure pH near the mucosal surface of anaesthetised COX-1 (-/-), COX-2 (-/-), or wild-type mice of the same genetic background. RESULTS: When the gastric mucosal surface was exposed and superfused (0.2 ml/min) with a weakly buffered saline solution (pH 3) containing the pH indicator Cl-NERF, the pH directly at the gastric surface and thickness of the pH gradient were similar in wild-type and COX-2 (-/-) mice, but COX-1 (-/-) mice had a significantly thinner pH gradient. Addition of indomethacin had minimal effects on the residual surface pH gradient in COX-1 (-/-) mice, suggesting no role for COX-2 in surface pH regulation. Whole stomach perfusion studies demonstrated diminished net alkali secretion in COX-1 (-/-) mice, and application of SC-560 or rofecoxib to wild-type mice and mutant mice confirmed that only COX-1 inhibition reduced alkali secretion. CONCLUSION: COX-1 is the dominant isoform regulating the normal thickness of the protective surface pH gradient in mouse stomach.

Magnetic resonance imaging of the dysplastic renal moiety and ectopic ureter.

PURPOSE: We determined the role of magnetic resonance imaging (MRI) in symptomatic children with clinically suspected and radiologically occult dysplastic renal moieties and ectopic ureters. MATERIALS AND METHODS: We reviewed clinical, imaging, cystoscopic, surgical and histological findings in 6 symptomatic children 1 to 15 years old with dysplastic renal moieties. RESULTS: After multiple conventional imaging studies failed to delineate urinary tract anatomy MRI provided detailed multiplanar images of dysplastic renal moieties that were diagnostic and predictive of subsequent intraoperative findings. Dysplastic upper pole moieties identified in 4 children were associated with ectopic ureters inserting into the vagina, prostatic urethra, bladder neck and bladder neck ureterocele in each. A solitary kidney with contralateral blind-ending ectopic ureters inserted into the bladder base in 2 cases. Pelvic cystic structures visualized by ultrasound in 3 patients were tortuous distal ureters on MRI. MRI specifically identified ureteral insertion sites that were not evident in 3 of the 5 patients who underwent cystoscopy. CONCLUSIONS: MRI may facilitate diagnosis, guide cystoscopy and aid in preoperative planning in children with poorly functioning renal moieties and ectopic ureters.

Incidence of contralateral inguinal hernia: a prospective analysis.

BACKGROUND/PURPOSE: Contralateral groin exploration in children with unilateral inguinal hernia is still controversial, particularly in infants. The authors have attempted to determine the age- and gender-stratified incidence of contralateral hernia and the necessity of routine bilateral procedures. METHODS: This is a prospective study of 656 patients during a 34-month period at a single institution. Patients with unilateral hernia underwent an ipsilateral procedure only, regardless of age, gestational age, or gender. Follow-up was 6 to 40 months (mean, 25.5 months). Chi-square analysis was used for intergroup comparison (P < .05 significant). RESULTS: Of 656 children, 108 (16.5%) presented with synchronous bilateral hernias. Bilateral inguinal hernia was significantly more common in premature infants (28.0%) and young children (33.8% if <6 months, 27.4% if <2 years). Of the remaining 548, a metachronous contralateral hernia developed in 48 (8.8%) at a median interval of 6 months (range, 4 days to 7 years). This incidence was 13 of 105 (12.4%) in infants less than 6 months of age, 20 of 189 (10.6%) in children less than 2 years of age, 8 of 54 (14.8%) in premature infants, 6 of 81 (7.4%) in girls, and 8 of 29 (27.6%) in children with an incarcerated hernia. In the latter group, P < .05, chi2 analysis. CONCLUSION: Routine contralateral inguinal exploration, without clinical evidence of a hernia, may be advisable in children with incarceration and possibly in premature infants. The low incidence of contralateral hernias in all other patients, regardless of gender or age, does not justify routine contralateral exploration.

New visualization techniques for in utero surgery: amnioscopy with a three-dimensional head-mounted display and a computer-controlled endoscope.

Endoscopic fetal surgery may reduce preterm labor associated with open hysterotomy but is partially limited by current visualization technology. We investigated a three-dimensional (3D) imaging system coupled to a head-mounted display (3D-HMD) and also employed a computer-controlled zoom endoscope for noninsufflated amnioscopy. Pregnant sheep were prepared in aseptic fashion for general anesthesia. Uterine access was obtained following maternal laparoscopy. A 10-mm zoom endoscope (Vista Medical Technologies, Carlsbad, CA) was used to examine the fetus and uterine contents. Fetal limbs were exteriorized for microsurgery. A new system (Vista Medical Technologies) was attached to an operative microscope, permitting projection of a 3D image via an HMD. The fetus and umbilical cord were inspected using the zoom endoscope, which changes the depth of focus under computer control. Basic manipulations of the fetus and cord were easily completed. Real-time 3D fetal imaging was accomplished. The added depth perception enabled detailed fetal and placental examination, fostering manipulation of the fetus and cord. The HMD was adjusted to fit several surgeons, permitting a natural operative posture. This unit has the capacity to display any video, CT, MR, or ultrasound image as a picture-in-picture. The success of minimally invasive fetal surgery is in part dependent on the development of video technologies capable of providing both magnification and optimal resolution. The zoom endoscope affords excellent visibility of multiple surgical targets without instrument repositioning. A 3D HMD system such as this provides greater anatomic detail and an appreciation of fetal movements that may make intrauterine procedures more feasible.

Temporary tracheal occlusion causes catch-up lung maturation in a fetal model of diaphragmatic hernia.

BACKGROUND: The lungs of infants born with diaphragmatic hernia are hypoplastic, immature, and surfactant-deficient. Tracheal occlusion in utero, which is being proposed as antenatal treatment of diaphragmatic hernia by promoting compensatory lung growth, decreases surfactant production as well, through loss of type II pneumocytes. The authors studied whether temporary tracheal occlusion might cause 'catch-up' lung growth and maturation, without negative effects of prolonged tracheal occlusion on the surfactant system. METHODS: Diaphragmatic hernia was created in time-dated fetal lambs (65 to 75 days). At 108 days, the trachea was occluded with an embolectomy catheter (DH + TO, n = 6). After day 14, the balloon was deflated. Six congenital diaphragmatic hernia (CDH) fetuses were left unobstructed (DH). For comparison, a group of fetuses without diaphragmatic hernia were subjected to prolonged tracheal ligation (TL; 4-week tracheal ligation, n = 3). Unoperated littermates (n = 8) were used as controls (CTR). All were killed near term. Lung tissue was processed for light and electron microscopy (computerized stereologic morphometry). Type II pneumocytes were identified with antisurfactant protein B antibody. RESULTS: Four animals in DH + TO and four in DH survived to term. Lung fluid volume (LFV) at 108 days was 5.2 +/- 4.4 mL in DH and 24.6 +/- 6.8 mL in controls (P < .05, Student t test). In DH + TO, LFV increased ninefold (to 48.3 +/- 13.3 mL) by 1 week postocclusion, suggesting accelerated lung growth. At term, lung weight to body weight ratio (LW/BW) was higher in TL (9.85% +/- 1.81%) than in CTR (3.55% +/- 0.56%; P < .05, analysis of variance); LW/BW and parenchymal volume tended to be greater in DH + TO than in DH, and air-exchanging parenchymal volume in DH + TO was similar to CTR (v a 50% reduction in DH), indicating some degree of hyperplasia after temporary occlusion. Pneumocyte II numerical density was decreased more than 10-fold in TL (60 +/- 22 v 826 +/- 324 in CTR, P < .001; it was slightly lower in DH + TO than in CTR, but individual type II pneumocyte cell volume was greater in the latter, and they appeared more mature than in DH (increased granulation by light microscopy, fewer glycogen granules, and abundant lamellar bodies by electron microscopy). Surfactant was also seen in the air spaces in DH + TO and CTR; it was absent in unobstructed CDH and in TL. CONCLUSIONS: Temporary tracheal occlusion in utero does not cause the dramatic decrease in type II pneumocytes seen after prolonged occlusion. Although only minimal increase in lung volume is seen in CDH, catch-up parenchymal growth and maturation occur, most notably in the surfactant-producing system.

Risk of nodal metastases at laparoscopic pelvic lymphadenectomy using PSA, Gleason score, and clinical stage in men with localized prostate cancer.

Laparoscopic pelvic lymph node dissection (LPLND) is a low-morbidity procedure used to stage prostate cancer accurately prior to definitive local therapy. To better select patients for LPLND, we reviewed the clinical features of 120 patients with clinically localized prostate cancer who underwent LPLND to define significant risk factors for nodal metastases. The age ranged from 43 to 79 years (mean 68). Serum prostate specific antigen (PSA) concentration ranged from 1.3 to 329 ng/mL, Gleason score ranged from 2 to 9, and clinical stage ranged from T1b to T3c. Nodal metastases were discovered in 15 patients (13%). Among men with a Gleason score > or = 7, 21% had nodal metastases (P = 0.004). A serum PSA > 20 ng/mL and clinical stage T1b, T2b, or greater also were statistically significant predictors of lymph node metastases (20% and 19%, respectively). In multivariate analysis, Gleason score significantly predicted nodal metastases when controlling for all other clinical measures. Therefore, LPLND is indicated for any patient with a Gleason score > or = 7, PSA > 20 ng/mL, and advanced clinical T stage, independently or in combination.

Antineoplastic agents. 362. Isolation and X-ray crystal structure of dibromophakellstatin from the Indian ocean sponge Phakellia mauritiana.

Bioassay-guided isolation procedures using human tumor cell lines led to isolation of dibromophakellstatin (4) from the Republic of Seychelles sponge Phakellia mauritiana. The isolation, X-ray crystal structure elucidation, absolute stereochemistry, and antineoplastic activity have been summarized. P. mauritiana was also found to contain dibromophakellin (1), debromohymenialosine (2), thymidine, deoxyuridine, and thymine.

Antineoplastic agents 338. The cancer cell growth inhibitory. Constituents of Terminalia arjuna (Combretaceae).

By means of bioassay-guided separation methods, the cancer cell growth inhibitory constituents residing in the bark, stem and leaves of the Mauritius medicinal plant Terminalia arjuna (Combretaceae) were examined. The cancer cell line active components were found to be gallic acid, ethyl gallate, and the flavone luteolin. Only gallic acid was previously known to occur in this plant. Luteolin has a well established record of inhibiting various cancer cell lines and may account for most of the rationale underlying the use of T. arjuna in traditional cancer treatments. Luteolin was also found to exhibit specific activity against the pathogenic bacterium Neisseria gonorrhoeae.

Antineoplastic agents, 325. Isolation and structure of the human cancer cell growth inhibitory cyclic octapeptides phakellistatin 10 and 11 from Phakellia sp.

The two new marine sponge (Phakellia sp., western Pacific Ocean) constituents, phakellistatin 10 [1] and 11 [2], were found to be cyclic octapeptides that significantly inhibited growth of the murine P-388 lymphocytic leukemia (ED50 values of 2.1 and 0.20 micrograms/ml, respectively) and human cancer cell lines. The structures were established based on results of extensive tandem ms/ms and high-field (500-MHz) 2D 1H- and 13C-nmr analyses. All of the amino acid units (except Trp, not determined) were found to correspond to the (S)-configuration.

Antineoplastic agents, 326. The stereochemistry of bastadins 8, 10, and 12 from the Bismarck archipelago marine sponge Ianthella basta.

An investigation of cancer cell-growth inhibitory constituents of the Papua New Guinea marine sponge Ianthella basta led to isolation of the C-6 hydroxybastadins 8 [1] 10 [2], and 12 [3]. The absolute stereochemistry (6S) of each bastadin (or its tetramethyl ester derivative) was determined by means of the Mosher-Trost method. Bastadins 10 [2] and 12 [3] were found to significantly inhibit the growth of a selection of human cancer cell lines. Bastadins 8, 10, and 12 inhibited growth of the Gram-positive opportunists Staphylococcus aureus and Enterococcus faecalis.

Antineoplastic agents. 278. Isolation and structure of axinastatins 2 and 3 from a western Caroline Island marine sponge.

The Republic of Palau marine sponge Axinella sp. was found to be an exceptionally productive source of cell growth inhibitory substances. The strongly antineoplastic polyether macrocyclic lactones halichondrin B (1) and homohalichondrin B (2) were isolated in 1.2 x 10(-6)% and 5.4 x 10(-7)% yields, respectively. In addition to axinastatin 1 (3), two new and cytostatic (GI50 values of 0.35 to 0.0072 microgram/mL against six human cancer cell lines) cycloheptapeptides designated axinastatins 2 (4) and 3 (5) were discovered in 1.4 x 10(-6)% and 1.25 x 10(-6)% yields. Structures were elucidated by high-resolution FABMS and tandem MS/MS techniques augmented by high-field (400 and 500 MHz) 2D-NMR spectral analyses. The absolute configurations were established by a combination of hydrolysis, derivatization, and chiral gas chromatographic methods.

Isolation and structure of the marine sponge cell growth inhibitory cyclic peptide phakellistatin 1.

A new cell growth inhibitory (P-388 murine leukemia ED50 7.5 micrograms/ml) cycloheptapeptide designated phakellistatin 1 was isolated from two Indo-Pacific sponges, Phakellia costata and Stylotella aurantium. Structural elucidation was accomplished utilizing high field nmr, amino acid analyses, and mass spectral techniques (fab, tandem ms/ms), followed by chiral gas chromatographic procedures for absolute configuration assignments (all S-amino acid units). By these methods phakellistatin 1 [1] was found to be cyclo (Pro-Ile-Pro-Ile-Phe-Pro-Tyr), and this assignment was finally confirmed by an X-ray crystal structure determination.

Studies of experimental cervical spinal cord transection. Part I: Hemodynamic changes after acute cervical spinal cord transection.

Two distinct and sequential patterns of hemodynamic alteration were observed after acute cervical spinal cord transection in anesthetized dogs. Interruption of the cord initially caused a 45% increase in mean arterial pressure (p less than 0.01), a 34% increase in systemic vascular resistance (p less than 0.05), and a 92% increase in left ventricular dp/dt (p less than 0.01), reflecting a generalized sympathetic response to trauma. Concomitant bradycardia and escape arrhythmias suggested relative parasympathetic hyperactivity. Resolution of the brief pressor response was followed by a second, more prolonged, period characterized by a fall in arterial pressure to 71% of control levels (p less than 0.05), a 16% decrease in systemic vascular resistance, and a 58.5% decrease in left ventricular dp/dt (p less than 0.01). These latter hemodynamic changes are consistent with sympathetic denervation and failure of regulatory mechanisms mediated by both alpha- and beta-adrenergic peripheral vascular and myocardial receptors.

The international standard for rolitetracycline.

An International Standard for Rolitetracycline has been established and the international unit of this antibiotic defined as the activity contained in 0.001004 mg of the international standard. The definition of the international unit was based on the results of a collaborative assay in which 8 laboratories in 6 different countries participated; a total of 133 assays were performed. The assay was in terms of the Working Standard of the USA Food and Drug Administration; mean potencies for individual laboratories varied within a range of only 2% of the mean for all assays although 7 different test organisms were used in both diffusion and turbidimetric assays. Individual assays, however, provided potencies that varied within a range of 40%.

The second international standard for chlortetracycline.

As supplies of the International Standard for Chlortetracycline were practically exhausted, it was replaced. The potency of the second international standard was defined on the basis of an international collaborative assay comprising 157 individual assays performed in 9 laboratories in 6 countries. The mean potencies obtained in the participating laboratories, although they varied by only 7%, were heterogeneous. However, the mean potency for all the laboratories combined did not differ significantly from that of the first international standard. The International Unit for chlortetracycline was therefore defined as the activity contained in 0.001 mg of the second international standard, corresponding to a potency of 1 000 IU/mg.