Umbilical venous catheter-induced hepatic hematoma in neonates.

Umbilical vessel catheterization is common practice for infants in the neonatal intensive care unit (NICU). Umbilical venous catheters (UVC) although very useful as a means of obtaining vascular access, do not come without risks. Here we will describe three separate cases of infants in the NICU who, during their course of treatment, were found to have hepatic masses attributed to UVC misplacement. Two of the cases presented incidentally and one presented acutely. We believe liver hematomas may be a more common complication of malpositioned UVCs than previously believed. An appreciation of the complications of malpositioned UVCs should alert clinicians to screen for potential complications and to ensure ideal line placement.

Neurological impairment in a surviving twin following intrauterine fetal demise of the co-twin: a case study.

It has been established that twin pregnancies are at an increased risk for complications, including the risk of morbidity or mortality for one or both of the infants. Cerebral palsy and other associated neurological deficits also occur at higher rates in twin pregnancies. This report examines two cases of intrauterine demise of one twin with subsequent survival of the co-twin. In both cases, the surviving infant suffered significant neurological sequelae. Impairments observed in these two cases include multicystic encephalomalacia and periventricular leukomalacia as well as the subsequent development of cerebral palsy. This case study explores the predisposing factors, incidence, pathophysiology, consequences, and future research implications of these findings.

NADPH oxidases and reactive oxygen species at different stages of chronic hypoxia-induced pulmonary hypertension in newborn piglets.

Recently, we reported that reactive oxygen species (ROS) generated by NADPH oxidase (NOX) contribute to aberrant responses in pulmonary resistance arteries (PRAs) of piglets exposed to 3 days of hypoxia (Am J Physiol Lung Cell Mol Physiol 295: L881-L888, 2008). An objective of the present study was to determine whether NOX-derived ROS also contribute to altered PRA responses at a more advanced stage of pulmonary hypertension, after 10 days of hypoxia. We further wished to advance knowledge about the specific NOX and antioxidant enzymes that are altered at early and later stages of pulmonary hypertension. Piglets were raised in room air (control) or hypoxia for 3 or 10 days. Using a cannulated artery technique, we found that treatments with agents that inhibit NOX (apocynin) or remove ROS [an SOD mimetic (M40403) + polyethylene glycol-catalase] diminished responses to ACh in PRAs from piglets exposed to 10 days of hypoxia. Western blot analysis showed an increase in expression of NOX1 and the membrane fraction of p67phox. Expression of NOX4, SOD2, and catalase were unchanged, whereas expression of SOD1 was reduced, in arteries from piglets raised in hypoxia for 3 or 10 days. Markers of oxidant stress, F(2)-isoprostanes, measured by gas chromatography-mass spectrometry, were increased in PRAs from piglets raised in hypoxia for 3 days, but not 10 days. We conclude that ROS derived from some, but not all, NOX family members, as well as alterations in the antioxidant enzyme SOD1, contribute to aberrant PRA responses at an early and a more progressive stage of chronic hypoxia-induced pulmonary hypertension in newborn piglets.

Polarizability of helium and gas metrology.

Using a quasispherical, microwave cavity resonator, we measured the refractive index of helium to deduce its molar polarizability A(epsilon) in the limit of zero density. We obtained (A(epsilon,meas) - A(epsilon,theory))/A(epsilon) = (-1.8 +/- 9.1) x 10(-6), where the standard uncertainty (9.1 ppm) is a factor of 3.3 smaller than that of the best previous measurement. If the theoretical value of A(epsilon) is accepted, these data determine a value for the Boltzmann constant that is only 1.8 +/- 9.1 ppm larger than the accepted value. Our techniques will enable a helium-based pressure standard and measurements of thermodynamic temperatures.

Selective inhibition of interleukin-4 gene expression in human T cells by aspirin.

Previous studies indicated that aspirin (acetylsalicylic acid [ASA]) can have profound immunomodulatory effects by regulating cytokine gene expression in several types of cells. This study is the first in which concentrations of ASA in the therapeutic range were found to significantly reduce interleukin (IL)-4 secretion and RNA expression in freshly isolated and mitogen-primed human CD4+ T cells. In contrast, ASA did not affect IL-13, interferon-gamma, and IL-2 expression. ASA inhibited IL-4, but not IL-2, promoter-driven chloramphenicol acetyltransferase expression in transiently transfected Jurkat T cells. The structurally unrelated nonsteroidal anti-inflammatory drugs indomethacin and flurbiprofen did not affect cytokine gene expression in T cells, whereas the weak cyclo-oxygenase inhibitor salicylic acid was at least as effective as ASA in inhibiting IL-4 expression and promoter activity. The inhibitory effect of ASA on IL-4 transcription was not mediated by decreased nuclear expression of the known salicylate target nuclear factor (NF)-kappaB and was accompanied by reduced binding of an inducible factor to an IL-4 promoter region upstream of, but not overlapping, the NF of activated T cells- and NF-kappaB-binding P1 element. It is concluded that anti-inflammatory salicylates, by means of a previously unrecognized mechanism of action, can influence the nature of adaptive immune responses by selectively inhibiting the expression of IL-4, a critical effector of these responses, in CD4+ T cells.

Modulation of epithelial morphogenesis and cell fate by cell-to-cell signals and regulated cell adhesion.

Cell-cell and cell-extracellular matrix (ECM) interactions control many developmental decisions of epithelial cell fate and morphogenesis. Protein tyrosine kinases are one class of regulatory molecules that have been implicated in the modulation of these processes. Several protein tyrosine kinases co-localize with cell-cell (cadherin) and cell-ECM (integrin) adhesion molecules at specific adhesion domains of epithelial cells. Protein tyrosine kinases may regulate epithelial development by modulating cell-cell and cell-ECM interactions and by relaying signals initiated by these interactions to other cellular components that determine cell structure and function.

pp60src tyrosine kinase modulates P19 embryonal carcinoma cell fate by inhibiting neuronal but not epithelial differentiation.

P19 embryonal carcinoma cells provide an in vitro model system to analyze the events involved in neural differentiation. These multipotential stem cells can be induced by retinoic acid (RA) to differentiate into neural cells. We have investigated the ability of several variant forms of the protein-tyrosine kinase (PTK) pp60src to modulate cell fate determination in this system. Normally, P19 cells are induced to differentiate along a neural lineage when allowed to form extensive cell-cell contacts in large multicellular aggregates during exposure to RA. Through analysis of markers of epithelial (keratin and desmosomal proteins) and neuronal (neurofilament) cells we have found that RA-induced P19 cells transiently express epithelial markers before neuronal differentiation. Under these inductive conditions, expression of pp60v-src or expression of the neuronal variant pp60c-src+ inhibited neuronal differentiation, and resulted in maintained expression of an epithelial phenotype. Morphological analysis showed that expression of pp60src PTKs results in decreased cell-cell adhesion during the critical cell aggregation stage of the neural differentiation procedure. The effects of pp60v-src on cell fate and cell-cell adhesion could be mimicked by direct modulation of Ca+(+)-dependent cell-cell contact during RA induction of normal P19 cells. We conclude that the neural lineage of P19 cells includes an early epithelial intermediate and suggest that tyrosine phosphorylation can modulate cell fate determination during an early cell-cell adhesion-dependent event in neurogenesis.

Palmitylation, sulfation, and glycosylation of the alpha subunit of the sodium channel. Role of post-translational modifications in channel assembly.

Antibodies to the alpha and beta 2 subunits and site-directed antibodies that distinguish alpha subunits of the RI and RII subtypes have been used to study the biosynthesis and assembly of sodium channels. The RII sodium channel subtype is preferentially expressed in rat brain neurons in primary cell culture. Post-translational processing of alpha subunits includes incorporation of palmityl residues in thioester linkage and sulfate residues attached to oligosaccharides. The incorporation of [3H] palmitate into alpha subunits is inhibited by tunicamycin, indicating that it occurs in the early stages of biosynthesis but after co-translational glycosylation. Mature alpha subunits are attached to beta 2 subunits through disulfide bonds within 1 h after synthesis and up to 30% can be specifically immunoprecipitated from the cell surface with antibodies against the beta 2 subunits by 4 h after synthesis. The remaining alpha subunits remain in an intracellular pool. The alpha subunits synthesized in the presence of castanospermine and swainsonine have reduced apparent size. Castanospermine prevents incorporation of approximately 81% of the sialic acid of the alpha subunit and inhibits sulfation but not palmitylation. Although inhibition of glycosylation with tunicamycin blocks assembly of functional sodium channels, castanospermine and swainsonine do not prevent the covalent assembly of alpha and beta 2 subunits or the transport of alpha beta 2 complexes to the cell surface, and sodium channels synthesized under these conditions have normal affinity for saxitoxin. Thus, the extensive processing and terminal sialylation of oligosaccharide chains during maturation of the alpha subunit is not essential. A kinetic model for biosynthesis, processing, and assembly of sodium channel subunits is presented.

Biosynthesis and processing of the alpha subunit of the voltage-sensitive sodium channel in rat brain neurons.

The sodium channel from rat brain is a complex of alpha (260 kd), beta 1 (36 kd), and beta 2 (33 kd) subunits. The alpha and beta 2 subunits are linked by disulfide bonds. The earliest biosynthetic precursor of the alpha subunit is a 203 kd core polypeptide with sufficient high-mannose carbohydrate chains to increase its apparent size to 224 kd. It is processed to 224 kd and 249 kd precursor forms containing complex carbohydrate chains before it achieves the mature size of 260 kd. Most newly synthesized alpha subunits are not disulfide-linked to beta 2 subunits, but remain as a metabolically stable pool of intracellular subunits. alpha subunits disulfide-linked to beta 2 are found preferentially at the cell surface. A possible role for this intracellular pool as a rate-limiting step in the regulation of the cell surface density and localization of sodium channels in developing neurons is proposed.

Reconstitution of calmodulin-sensitive adenylate cyclase from bovine brain with phosphatidylcholine liposomes.

A partially purified calmodulin (CaM)-sensitive adenylate cyclase from bovine cerebral cortex was reconstituted with a series of phosphatidylcholine liposomes having variable fatty acid composition. The enzyme was successfully associated with dimyristoyl, dipalmitoyl, distearoyl, and dioleoylphosphatidylcholine liposomes. The specific activity of the enzyme in the various liposomes varied over a 4.6-fold range indicating some degree of specificity for fatty acid composition. The adenylate cyclase-liposome preparation retained sensitivity to both CaM and 5'-guanylylimidodiphosphate (GppNHp). Arrhenius plots of enzyme activity in the four different liposome preparations all exhibited a pronounced discontinuity at 30 degrees C +/- 2, even though the bulk-phase thermal transition points for the liposomes varied from -20 to 54 degrees C. Fluorescence anisotropy studies of reconstituted liposome systems illustrated that incorporation of protein did not alter the normal-phase transition point of these lipids. Since Arrhenius plots of the enzyme in Lubrol PX, prior to reconstitution with lipids, were strictly linear, it is concluded that the breaks at 30 degrees C may be the effect of a local enzyme-phospholipid environment. It appears that this adenylate cyclase is not particularly sensitive to phase transitions of the bulk lipid phase. The phospholipid reconstituted enzyme system appears suitable for examination of the influence of lipids on the CaM-sensitive adenylate cyclase.

On the failure of calmodulin to activate Ca2+ pump ATPase of dog red blood cells.

Ca2+-pump ATPase activities of membranes isolated from human and dog RBCs were compared under a variety of conditions. Specific activity of the dog enzyme was less than that of human. Unlike the human enzyme, the dog Ca2+-pump ATPase was not stimulated by exogenously added calmodulin (CaM) or oleate. The Ca2+ dependence of the dog Ca2+-pump ATPase resembled that of the CaM-activated form of the human enzyme. Cross-linking of Azido-125I-CaM to dog RBC membranes did not label a Ca2+-pump ATPase of molecular weight similar to that found in human RBC membranes. It is suggested that the Ca2+-pump ATPase in isolated dog RBC membranes exists in an activated state, not due to endogenous CaM, but possibly due to partial proteolysis.

Glycosylation is required for maintenance of functional sodium channels in neuroblastoma cells.

Experimental conditions were established under which tunicamycin inhibits glycosylation by 80-90% but reduces protein biosynthesis by only 10-20% in cultured neuroblastoma cells. Growth in the presence of tunicamycin (1 micrograms/ml) reduces the number of sodium channels, as measured by high affinity saxitoxin (STX) binding to 20-28% of control values over a 60-h period without affect on the KD for STX. Neurotoxin-activated 22Na+ influx mediated by the sodium channel was similarly reduced without affect on the KD for batrachotoxin. Comparison of STX binding by intact cells or homogenates showed that tunicamycin reduces cell surface STX receptors without accumulation of an intracellular pool of binding sites. Tunicamycin caused a similar reduction in cell surface STX receptors in the presence of the lysosomal inhibitor chloroquine, suggesting that its action is not entirely due to acceleration of sodium channel degradation. The action of tunicamycin is at least partially reversible. After washout, STX receptors appear at an initial rate of approximately 1900/cell/h. Protein synthesis is required for the appearance of new sodium channels. After inhibition of sodium channel biosynthesis by either cycloheximide or tunicamycin, the number of high affinity STX receptor sites is reduced with a half-time of 26 h. Thus, at steady state, neuroblastoma cells which contain 50,000 +/- 15,000 STX receptors degrade and replace 1330 +/- 400 STX receptor sites/h. Our results show that glycosylation is an essential process in the maintenance of the normal steady state of biosynthesis and degradation of sodium channels.

Comparative protein quality as measured by human and small animal bioassays of three lines of winter wheat.

Incomplete information on factors contributing to apparent protein quality and to value of food products as sources of protein and how these factors interact necessitate the use of bioassay procedures. Ideally bioassay procedures should be done using the animal species for which the protein is intended. Practical considerations dictate the use of small animal bioassay rather than human bioassays for routine use in protein product evaluation. To be of real value for routine use in protein product evaluation. To be of real value for assays of food products designed for human use, animal bioassays must accurately predict human performance. Surprisingly little information is available on this topic. In the current project three Nebraska winter wheats of similar genetic backgrounds were evaluated for protein value and for value of the wheats as sources of proteins. Chemical, weanling mouse, adult human and growing human bioassay techniques were employed. Rankings of the grains were similar regardless of species used for protein quality evaluations. Similar rankings were found regardless of species used for protein quality/quantity evaluations. However, ranking varied between methods designed to evaluate protein quality and those designed to measure protein quality/quantity interrelationships. The results stress the importance of matching appropriate methodology with information desired. In a latter project, wheats of dissimilar genetic background were not as uniformily evaluated. This suggests that other factors known to affect protein quality and value were more variable in these wheats.