Pigmented epithelium sustains cell proliferation and decreases expression of opsins and acetylcholinesterase in reaggregated chicken retinospheroids

We investigated the effect of the retinal pigmented epithelium on cell proliferation and differentiation in rosetted retinospheroids, which are retina-like spheres reaggregated in the complete absence of retinal pigmented epithelium from dissociated retinal cells of 6-day-old chick embryos in a rotation culture system. In spheroids raised in the absence of retinal pigmented epithelium (controls), acetylcholinesterase was expressed in cells of an inner nuclear-like layer and their neuropil matrices. Moreover, the ratio between rods and cones was found to be approximately normal throughout the spheroid. When spheroids were cultured in the presence of retinal pigmented epithelium monolayers, cell proliferation in spheroids as determined by BrdU labelling was significantly increased and extended for 1 week, while acetylcholinesterase protein levels and specific activities in homogenates were decreased to approximately 30%. At the same time, opsin immunoreactivity was completely suppressed within the spheroid and appeared slowly in cells around its periphery; i.e. the proportion of rhodopsin-positive cells decreased from 14 to 3%. This study reveals that the retinal pigmented epithelium in vitro sustains cell proliferation but inhibits the differentiation of acetylcholinesterase-positive cells and of photoreceptors.     

Unfolding and inactivation of Penaeus penicillatus acid phosphatase during denaturation by guanidine hydrochloride

Neuromuscular terminals of a single motoneuron to four muscles (CPV7a, GM5a, CV2, and CV3) in the stomach of the blue crab Callinectes sapidus showed structural evidence for the exocytotic release of dense-core vesicles exclusively at synapses. The primary evidence was the appearance of dense cores in the synaptic cleft, accompanied by indentations of the presynaptic or postsynaptic membrane. In their simplest form, these consisted of an omega-shaped figure of the presynaptic membrane enclosing one dense core, denoting release of a single dense-core vesicle. A larger indentation of the presynaptic membrane enclosing several dense cores denoted multiple release. A more complex form of multiple release was where the presynaptic membrane was normal, but the postsynaptic membrane elaborated into a sac projecting into the granular sarcoplasm and filled with dense cores. The postsynaptic sac in some instances was compressed into a thin, fingerlike extension, which lacked dense cores and, at its distal end, separated into small cisternae, suggesting a mechanism for membrane recycling. Profiles depicting single and multiple releases of dense-core vesicles were found more frequently at neuromuscular terminals that release relatively large amounts of transmitter with a single stimulus, such as CV2 and CV3, compared to those releasing smaller amounts, such as CPV7a and GM5a. The disparity in release sites among the four muscles of this single motor unit and the fact that many of the multiple-release figures were closely adjacent to the active zones for transmitter release suggest a possible modulatory role for dense-core vesicles in synaptic transmission. Such modulation may be long lasting, as implied by the postsynaptic sacs, which may permit prolonged release of the contents of their dense cores into the synaptic cleft. This is in keeping with the functional role of these stomach muscles, which is to be continuously active for long periods of time.     

Midline dorsal appendages

A case of a mature posterior cervical teratoma resembling two fingers and a case of a lumbosacral limb are presented. These are at least the seventh and eighth reported paraspinous or occipital teratomas with recognizable extremity development. Both anomalies had intradural extension and required exploration to decrease the likelihood of future spinal cord tethering. Preoperative evaluation, surgical technique, postoperative follow-up, and review of the literature are presented.     

Performance evaluation of a whole-body PET scanner using the NEMA protocol. National Electrical Manufacturers Association

This study evaluates the performance of the newly developed high-resolution whole-body PET scanner ECAT EXACT HR+. METHODS: The scanner consists of four rings of 72 bismuth germanate block detectors each, covering an axial field of view of 15.5 cm with a patient port of 56.2 cm. A single block detector is divided into an 8 x 8 matrix, giving a total of 32 rings with 576 detectors each. The dimensions of a single detector element are 4.39 x 4.05 x 30 mm3. The scanner is equipped with extendable tungsten septa for two-dimensional two-dimensional measurements, as well as with three 68Ge line sources for transmission scans and daily quality control. The spatial resolution, scatter fraction, count rate, sensitivity, uniformity and accuracy of the implemented correction algorithms were evaluated after the National Electrical Manufacturers Association protocol using the standard acquisition parameters. RESULTS: The transaxial resolution in the two-dimensional mode is 4.3 mm (4.4 mm) in the center and increases to 4.7 mm (4.8 mm) tangential and to 8.3 mm (8.0 mm) radial at a distance of r = 20 cm from the center. The axial slice width measured in the two-dimensional mode varies between 4.2 and 6.6 mm FWHM over the transaxial field of view. In the three-dimensional mode the average axial resolution varies between 4.1 mm FWHM in the center and 7.8 mm at r = 20 cm. The scatter fraction is 17.1% (32.5%) for a lower energy discriminator level of 350 keV. The maximum true event count rate of 263 (345) kcps was measured at an activity concentration of 142 (26.9) kBq/ml. The total system sensitivity for true events is 5.7 (27.7) cps/Bq/ml. From the uniformity measurements, we obtained a volume variance of 3.9% (5.0%) and a system variance of 1.6% (1.7%). The implemented three-dimensional scatter correction algorithm reveals very favorable properties, whereas the three-dimensional attenuation correction yields slightly inaccurate results in low- and high-density regions. CONCLUSION: The ECAT EXACT HR+ has an excellent, nearly isotropic spatial resolution, which is advantageous for brain and small animal studies. While the relatively low slice sensitivity may hamper the capability for performing fast dynamic two-dimensional studies, the scanner offers a sufficient sensitivity and count rate capacity for fully three-dimensional whole-body imaging.     

Acute aortic dissection complicating pregnancy

BACKGROUND: Acute aortic dissection occurring during pregnancy represents a lethal risk to both the mother and fetus. Our purpose was to study the prevalence, treatments, and outcome of this rare problem and to suggest therapeutic guidelines. METHODS: During the past 12 years, 6 pregnant women were admitted with an acute aortic dissection. Four had a type A and 2 had a type B dissection (Stanford classification). RESULTS: Two of the 4 patients with a type A dissection underwent a combined emergency operation consisting of first cesarean section and then ascending aortic repair. Cesarean section was carried out 5 days after the emergency procedure on the aorta in the third patient, and 16 weeks later in the fourth patient. All 4 fetuses were delivered alive. One fetus died 6 days later, but the other 3 are alive and well at long-term follow-up. Of the 2 patients with a type B dissection, 1 was operated on for celiac ischemia; the other was treated medically. In both cases the fetus died in utero. There were no maternal deaths in either group. CONCLUSIONS: Cesarean section with concomitant aortic repair is recommended for pregnant women with a type A dissection, depending on the gestational age. The maternal hemodynamic status will determine the sequence of the two procedures. Medical treatment is advised for patients with a type B dissection, but surgical repair is indicated if complications such as bleeding or malperfusion of major side branches occur.     

Pharmacokinetics and safety of tobramycin after once-daily administration in patients with cystic fibrosis

STUDY OBJECTIVE: Tobramycin is commonly used to treat respiratory tract infections in patients with cystic fibrosis. We designed a study to determine the pharmacokinetics and safety of once-daily dosing of tobramycin in this population. DESIGN: Multiple blood samples were collected from each patient, and serum concentrations of tobramycin were determined by a fluorescence polarization immunoassay. Blood urea nitrogen and serum creatinine levels were measured every 2 to 3 days, and audiometric evaluations were performed at the start and end of therapy. MEASUREMENTS AND RESULTS: Eighteen patients (mean age, 24.6 years) received tobramycin at doses of 7 to 15 mg/kg/d as a single-dose infusion over 20 min. The maximum serum concentration of tobramycin ranged from 40.1 to 64.6 mg/L. A mean dose of 11.9+/-1.9 mg/kg was needed to obtain a theoretical mean peak serum concentration of 42.4+/-4.5 mg/L. The mean total body clearance, apparent volume of distribution, and elimination half-life was 1.7+/-0.4 mL/min/kg, 0.27+/-0.06 L/kg, and 1.8+/-0.3 h, respectively. The period of time that the serum concentration exceeded eight times the theoretical minimum inhibitory concentration of 1 mg/L ranged from 2.1 to 4.4 h, which was nearly five times longer compared with the use of divided daily doses in the same patients during previous hospitalizations. No nephrotoxicity, ototoxicity, or adverse effects occurred in any patient. CONCLUSION: Based on our data, tobramycin may be used safely in once-daily doses to treat exacerbations of respiratory tract infections in patients with cystic fibrosis.     

Lung transplantation in children

The first lung transplantation in the Netherlands was carried out in 1990. Since, the operation has been performed in over one hundred adults and two children (up to 16 years) with chronic respiratory insufficiency. Most lung transplantations in children are performed because of cystic fibrosis. After referral, the patient is given information about the operation and the pre- and postoperative periods, and the transplantation team advises on the time of screening. Some one-third of the children referred ultimately undergo transplantation; the others refrain from the operation, are rejected or die during the waiting period. Owing to a shortage of donor lungs the waiting period for transplantation may last over one year. A possible but controversial solution is donation of a pulmonary lobe by a close relative. Undergoing lung transplantation places a heavy (psychic) burden on the patient and his parents. Every patient after lung transplantation goes through an average of two or three periods of acute rejection. Chronic rejection occurs in 20-40%. Other complications are connected to medication (infections, renal dysfunction) or to the underlying disease (diabetes mellitus in cystic fibrosis). The percentages of survival for 1, 2 and 4 years are approx. 60-70, 55-60 and 30-50, dependent in part on the type of lung transplantation (unilateral, bilateral or combined heart-lung transplantation). A reasonable condition of the patient owing to timely referral is a positive prognostic factor.     

Heterologous expression of rat epitope-tagged histamine H2 receptors in insect Sf9 cells

1. Rat histamine H2 receptors were epitope-tagged with six histidine residues at the C-terminus to allow immunological detection of the receptor. Recombinant baculoviruses containing the epitope-tagged H2 receptor were prepared and were used to infect insect Sf9 cells. 2. The His-tagged H2 receptors expressed in insect Sf9 cells showed typical H2 receptor characteristics as determined with [125I]-aminopotentidine (APT) binding studies. 3. In Sf9 cells expressing the His-tagged H2 receptor histamine was able to stimulate cyclic AMP production 9 fold (EC50=2.1+/-0.1 microM) by use of the endogenous signalling pathway. The classical antagonists cimetidine, ranitidine and tiotidine inhibited histamine induced cyclic AMP production with Ki values of 0.60+/-0.43 microM, 0.25+/-0.15 microM and 28+/-7 nM, respectively (mean+/-s.e.mean, n=3). 4. The expression of the His-tagged H2 receptors in infected Sf9 cells reached functional levels of 6.6+/-0.6 pmol mg(-1) protein (mean+/-s.e.mean, n=3) after 3 days of infection. This represents about 2 x 10(6) copies of receptor/cell. Preincubation of the cells with 0.03 mM cholesterol-beta-cyclodextrin complex resulted in an increase of [125I]-APT binding up to 169+/-5% (mean+/-s.e.mean, n=3). 5. The addition of 0.03 mM cholesterol-beta-cyclodextrin complex did not affect histamine-induced cyclic AMP production. The EC50 value of histamine was 3.1+/-1.7 microM in the absence of cholesterol-beta-cyclodextrin complex and 11.1+/-5.5 microM in the presence of cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). Also, the amount of cyclic AMP produced in the presence of 100 microM histamine was identical, 85+/-18 pmol/10(6) cells in the absence and 81+/-11 pmol/10(6) cells in the presence of 0.03 mM cholesterol-beta-cyclodextrin complex (mean+/-s.e.mean, n=3). 6. Immunofluorescence studies with an antibody against the His-tag revealed that the majority of the His-tagged H2 receptors was localized inside the insect Sf9 cells, although plasma membrane labelling could be identified as well. 7. These experiments demonstrate the successful expression of His-tagged histamine H2 receptors in insect Sf9 cells. The H2 receptors couple functionally to the insect cell adenylate cyclase. However, our studies with cholesterol complementation and with immunofluorescent detection of the His-tag reveal that only a limited amount of H2 receptor protein is functional. These functional receptors are targeted to the plasma membrane.     

Increased levels of plasma cholesteryl ester hydroperoxides in patients with subarachnoid hemorrhage

The pathophysiology of subarachnoid hemorrhage (SAH) may involve free radical production and lipid peroxidation. We examined plasma levels of cholesteryl ester hydroperoxides (CEOOH) and antioxidants in 25 patients with SAH, and 10 neurologic controls with lacunar stroke. Patients with SAH had significantly increased plasma levels of CEOOH, which peaked on day 5 after the ictus. Concentrations of CEOOH were significantly increased, and ascorbic acid concentrations were significantly decreased in patients who developed vasospasm compared with patients without vasospasm. Increased levels of CEOOH were associated with increased mortality and correlated with clinical outcome scales. These results implicate oxidative stress in the pathogenesis of SAH and suggest that measurements of CEOOH in plasma may be useful both prognostically as well as in monitoring therapeutic interventions.     

Phospholipase A2 in human placental serum

Intervillous blood was collected from term placentae at delivery, and sera were tested for phospholipase A2 under various experimental conditions. Enzyme activity was found to develop upon extended storage in the cold or at 37 degrees C. The enzyme is reversibly inhibited by dithiothreitol, requires Ca++ ions for activity, and tolerates various detergents. The apparent molecular weight is 42 kDa. In all these parameters the serum enzyme behaves similar to the 42 kDa phospholipase A2 which we recently purified to homogeneity from thoroughly washed placental tissue. Serum phospholipase A2 appears to be generated by proteolytic processing from a slightly larger inactive precursor which was detected immunochemically. Most likely this protein originates from fetal cells and may be released by membrane damage. We conclude that both placental serum and tissue harbour a novel type of phospholipase A2 which is distinct from cytosolic and secretory phospholipases A2. Preference for arachidonate containing substrate suggests a role in eicosanoid production within gestational tissues.