Lipid labeling with32P-orthophosphate and14C-acetate in marine copepods

Freshly collectedCalanus pacificus were maintained in sea water containing 25 μCi/ml [³²P]orthophosphate or 1 μCi/ml [¹⁴C]acetate at 10 C for 24 hr. The animals took up label from the environment and incorporated it into various lipid fractions. After incubation with [¹⁴C]acetate the order of specific activity of the different lipid classes was: phospholipids > free fatty acids > wax esters > triglycerides. Argentation thin layer chromatography of the fatty acid methyl esters showed that ca. 50% of the activity was in saturated fatty acids and 34% in polyunsaturated acids. When the animals were exposed to [³²P]orthophosphate, lysophosphatidyl choline became most heavily labeled, followed by lysophosphatidyl ethanolamine, sphingomyelin, phosphatidyl ethanolamine, and phosphatidyl choline. Comparison of the data obtained with those available for decapods and mammals revealed striking similarities between these phylogenetically distant groups. It is believed that labeling the lipids of marine and freshwater planktonic crustaceans in this way will provide much information about the metabolism of lipids in these organisms.     

Changes in the size and docosahexaenoic acid content of adipocytes during chick embryo development

The development of adipose tissue in the chick embryo was investigated using two groups of fertile eggs which differed by 1.7-fold in their initial yolk lipid levels. The triacylglycerol content of the subcutaneous adipose depot in both groups increased dramatically from day 12 of the 21-day embryonic period, attaining a maximal value just prior to hatching. During this period, the amount of triacylglycerol deposited in the adipose tissue was very highly correlated with the amount of lipid transferred from the yolk. The triacylglycerol content of the depot was also dependent on the initial yolk lipid content. During the hatching period, the amount of adipose triacylglycerol remained approximately constant in the group with the higher initial yolk lipid content but, in the case of the group with the lower initial yolk lipid levels, decreased by approximately 25%. The size distribution of adipocytes isolated from the tissue was determined by computerized image analysis microscopy. The mean adipocyte diameter increased from approximately 6 to 35 μm between days 12 and 19, irrespective of the initial yolk content, although development within the eggs with the lower initial yolk content resulted in a decrease in cell size over the hatching period. Both the triacylglycerol and phospholipid fractions of the isolated adipocytes contained substantial proportions (approximately 6%, w/w) of docosahexaenoic acid (DHA) at days 12 and 14, and lower levels of this fatty acid at the later stages. The amount (mg/depot) of DHA in adipose triacylglycerol decreased dramatically over the hatching period. The amount (mg/brain) of DHA in brain phospholipid increased by more than 5-fold between day 12 of development and hatching. A possible explanation for the data may be that DHA is preferentially mobilized from adipose tissue in order to deliver the fatty acid to the developing neural tissues in a form suitable for uptake.     

Minimum cost design of laterally loaded welded rectangular cellular plates

Material and fabrication costs are included in the cost function. The fabrication cost is calculated by three formulae relating to the preparation, welding and additional costs. The design constraints are related to bending stresses, the local buckling of ribs due to bending and shear and to the limitation of the plate thicknesses. The local buckling of the compressed face plate elements is considered by an effective width calculation. In the numerical examples, the variables are the plate dimensions and the numbers of ribs in two directions. The optimization is carried out for steel Fe 360 and Fe 510 and for various values of the fabrication cost factor. The computations are performed by using the backtrack discrete combinatorial method, Rosenbrock's Hillclimb method and the FSQP method developed by Zhou and Tits (1992), and the results are compared with each other.Partly presented at the international conference Structural Optimization '93, Rio de Janeiro, August 2–6, 1993.     

Real-time patch-cram detection of intracellular cGMP reveals long-term suppression of responses to NO and muscarinic agonists

Cyclic GMP (cGMP) is a crucial intracellular messenger in neuronal, muscle, and endocrine cells. The intracellular concentration of cGMP is regulated by various neurotransmitters, including acetylcholine (ACh) and nitric oxide (NO). While much is known about the biochemical steps leading to cGMP synthesis, little is known about cGMP kinetics in intact cells. Here, we use "patch-cramming," in which an excised, inside-out membrane patch containing cyclic nucleotide-gated ion channels is used as a biosensor, to obtain the first real-time measurements of cGMP in intact cells. Patch-cramming experiments on neuroblastoma cells show that both muscarinic agonists and NO rapidly elevate cGMP. NO elicits cGMP responses repeatedly without decrement, whereas responses to muscarinic agonists exhibit a profound and prolonged desensitization. Remarkably, muscarinic agonists also cause long-term (>30 min) suppression (LTS) of cGMP responses elicited by NO. Biochemical measurements reveal that rat sympathetic neurons also exhibit LTS of cGMP, suggesting that LTS is a widespread mechanism that may contribute to synaptic plasticity.     

Binding and lysing of blood clots using MRX-408

RATIONALE AND OBJECTIVES: A thrombus-specific ultrasound contrast agent, MRX-408, has been developed recently. This agent consists of phospholipid-coated microbubbles with a ligand capable of targeting the GPIIb/IIIa receptor, thereby allowing the microbubbles to bind with thrombi rich in activated platelets. In vitro and in vivo animal experiments have been conducted to examine imaging enhancement and sonothrombolysis using this agent compared with a nontargeted agent. METHODS: For clot binding, blood-smeared slides were incubated with microbubbles and examined under a light microscope. Change in backscatter signals from the blood clots after binding was examined by both an ultrasound scanner and two single-element transducers arranged in a transmitter-receiver pair. For clot lysis, either 1-MHz or 20-KHz ultrasound was used to enhance the lysing effects of MRX-408 with or without urokinase. RESULTS: Evidence of binding was demonstrated under a microscope. In vitro experiments showed that the "acoustic signature", or properties, of blood clots changed after binding. Clots became more echogenic and nonlinear. In vivo fundamental ultrasound imaging confirmed that as a result of binding, blood clots were more visible, the area of detection was improved, and shadowing behind clots was more noticeable. Under 1-MHz ultrasound and 30 minutes of treatment, lysis efficiency reached 34% with MRX-408, whereas there was no visible clot lysis with saline. CONCLUSION: The results of these preliminary studies show that as a contrast agent, MRX-408 enhanced clots under ultrasound imaging and facilitated sonothrombolysis with or without thrombolytic drugs.     

A Secure Grid Medical Data Manager Interfaced to the gLite Middleware

The medical community is producing and manipulating a tremendous volume of digital data for which computerized archiving, processing and analysis is needed. Grid infrastructures are promising for dealing with challenges arising in computerized medicine but the manipulation of medical data on such infrastructures faces both the problem of interconnecting medical information systems to Grid middlewares and of preserving patients’ privacy in a wide and distributed multi-user system. These constraints are often limiting the use of Grids for manipulating sensitive medical data. This paper describes our design of a medical data management system taking advantage of the advanced gLite data management services, developed in the context of the EGEE project, to fulfill the stringent needs of the medical community. It ensures medical data protection through strict data access control, anonymization and encryption. The multi-level access control provides the flexibility needed for implementing complex medical use-cases. Data anonymization prevents the exposure of most sensitive data to unauthorized users, and data encryption guarantees data protection even when it is stored at remote sites. Moreover, the developed prototype provides a Grid storage resource manager (SRM) interface to standard medical DICOM servers thereby enabling transparent access to medical data without interfering with medical practice.     

Collection and transfusion of blood and blood components in the United States, 1989

A poor response to L-DOPA in addition to parkinsonian, cerebellar, and autonomic signs is commonly regarded as indicative of clinical multiple system atrophy (MSA). We compared the motor response to a single oral administration of 250 mg L-DOPA/25 mg carbidopa in eight MSA patients and eight Parkinson's disease (PD) patients with the "on-off" phenomenon, evaluating L-DOPA peripheral pharmacokinetics. Motor response was consistently good in all PD patients, but only four MSA patients had a (moderate) response. Pharmacokinetic parameters did not differ between the groups. The varying extent of putaminal damage could be responsible for the differing motor response to L-DOPA in MSA patients.     

Age incidence of meningococcal infection England and Wales, 1984-1991

Gene transfer with vectors derived from murine retroviruses is restricted to cells which are proliferating and synthesizing DNA at the time of infection. This suggests that retroviral-mediated gene transfer might permit targeting of gene integration into malignant cells in organs composed mainly of quiescent nonproliferating cells, such as in the brain. Accordingly, selective introduction of genes encoding for susceptibility to otherwise nontoxic drugs ("suicide" genes) into proliferating brain tumors may be used to treat this cancer. We investigated the efficacy and dynamics of in vivo transduction of growing brain tumors with the herpes simplex-thymidine kinase gene followed by administration of the antiviral drug ganciclovir. Ganciclovir is phosphorylated by thymidine kinase to toxic triphosphates that interfere with DNA synthesis, resulting in the preferential death of the transduced tumor cells. Rats inoculated with 4 x 10(4) 9L gliosarcoma cells into the frontal lobe were treated 7 days later with an intratumoral stereotaxic injection of murine fibroblasts (NIH 3T3 cells) that were producing a retroviral vector containing the herpes simplex-thymidine kinase gene. Controls received vector producer and nonproducer NIH 3T3 cell lines containing the Escherichia coli lacZ (beta-galactosidase) gene as well as nonproducer NIH 3T3 cells containing the thymidine kinase gene. The animals were rested for 7 days to allow time for in situ transduction of the proliferating tumor cells with the herpes-thymidine kinase retroviral vector. The animals were then treated with ganciclovir, 15 mg/kg i.p. twice a day for 14 days. Gliomas receiving an injection of 3-5 x 10(6) thymidine kinase producer cells regressed completely in 23 of 30 rats given ganciclovir therapy, while 25 of 26 control rats developed large tumors. Intratumoral injection of a lower concentration of thymidine kinase vector producer cells (1.8 x 10(6)) resulted in a lower frequency of tumor regression (5 of 13 rats). To estimate the efficiency of in vivo gene transfer, 9L brain tumors were given injections of 5 x 10(6) beta-galactosidase vector producer cells. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranaside staining revealed maximal staining of beta-galactosidase within the tumor 7-14 days after injection of the vector producer cells. In vivo transduction rates in harvested tumors ranged from 10 to 70%. There was no evidence of transduction of the surrounding normal neural tissue. Occasional blood vessel endothelial cells within or adjacent to the tumor were observed to be 5-bromo-4- chloro-3-indolyl-beta-D-galactopyranaside positive.(ABSTRACT TRUNCATED AT 400 WORDS)     

Activation of the mouse heart adenosine 5',5"'-P1-P4-tetraphosphate receptor

We have previously demonstrated that mouse brain membrane fractions have a specific, saturable receptor for diadenylated nucleotides. Binding is specific for two adenosines, and the length of the phosphate bridge is critical, with four phosphates being optimal [Hilderman et al. (1991) J. Biol. Chem. 266, 6915-6918]. In this report, we demonstrate that adenosine 5',5"'-P1,P4-tetraphosphate (Ap4A) binding to its receptor is dependent upon an activation step that requires divalent cations and a serine protease. Monoclonal antibodies (Mabs) are identified that inhibit Ap4A binding to its membrane receptor. These antibodies recognize a 212-kDa membrane protein. However, SDS-PAGE analysis of Ap4A cross-linked to membrane fractions reveals that Ap4A is not attached to the 212-kDa peptide but to a 30-kDa polypeptide. Appearance of the 30-kDa polypeptide is dependent on the activation step, and one of the inhibitory antibodies blocks its appearance. We suggest that the protease-dependent processing step involves cleavage of the 212-kDa component with the appearance of an active 30-kDa receptor.     

Relative importance of growth hormone and sex steroids for the growth at puberty of trunk length, limb length, and muscle width in growth hormone-deficient children

We have followed the growth of stature, sitting height, skinfolds, muscle widths measured radiologically, and skeletal maturity in growth hormone-deficient patients in whom hGH was given and withheld in alternating three-month periods throughout puberty (referred to as "off-hGH" and "on-hGH" periods). Six boys and four girls had true isolated GH deficiency and developed puberty spontaneously. Two boys had gonadotrophin deficiency plus GH deficiency, and five boys had multiple deficiencies; in these boys the signs of puberty were induced by hormone treatment. Boys with true isolated deficiency grew about two-thirds as much in height in the off-hGH periods as in the on-hGH periods; their total gain in height during the adolescent spurt would have been about 20 cm, instead of 30 cm, if hGH had been discontinued at the beginning of puberty. The effect of hGH was entirely on growth in leg-length, however, which virtually ceased during the off-hGH periods. Growth in sitting height altered little when hGH was withdrawn. Growth in limb muscles, however, was GH dependent throughout puberty; during the majority of periods when hGH was withheld, muscle was actually lost; this occurred in the boys who were receiving large doses of testosterone as well as in those producing their own normal amounts. Subcutaneous fat diminished when hGH was given and increased when it was withdrawn; this occurred independently of administration of testosterone. There was little evidence that growth of pubic and axillary hair progressed faster during on-hGH periods, except perhaps in patients with multiple deficiencies. There was some evidence, however, that bone age progressed less rapidly during on-hGH periods than during off-hGH periods in the patients with isolated deficiency. The results in the girls agreed with those in boys so far as stature was concerned, but the relationship with sitting height and leg length appeared to be different; the reasons for this are discussed. We conclude that all children with GH deficiency should continue on treatment with hGH throughout puberty, ideally until growth ceases.