Antagonistic roles for phospholipase D activities in B cell signaling: while the antigen receptors transduce mitogenic signals via a novel phospholipase D activity, phosphatidylcholine-phospholipase D mediates antiproliferative signals

Cross-linking of the Ag receptors on B cells induces DNA synthesis and proliferation. Butanol trap experiments suggest that one or more phospholipase D activities play a key role in this process. Although phosphatidylcholine-phospholipase D has been shown to play a central role in the transduction of proliferative responses for a wide variety of calcium-mobilizing receptors, we show that the Ag receptors are not coupled to this phospholipase. In addition, phosphatidylcholine-phospholipase D is not stimulated under conditions that mimic T cell-dependent B cell activation. In contrast, ATP, which inhibits surface Ig (sIg)-mediated DNA synthesis in murine B cells via P2-purinoceptors, activates phosphatidylcholine-phospholipase D. Phosphatidylcholine-phospholipase D is therefore associated with antiproliferative signal transduction in mature B cells, but it does not transduce early signals associated with sIg-mediated growth arrest or apoptosis in immature B cells. Mitogenic stimulation of sIg is, however, coupled to a novel nonphosphatidylcholine-hydrolyzing phospholipase D activity. The resultant sIg-generated phosphatidic acid, unlike the phosphatidylcholine-derived phosphatidic acid generated via the purinoceptors, is converted to diacylglycerol. These data provide the first evidence that while the novel sIg-coupled phospholipase D and resultant diacylglycerol generation may play a role in B cell survival and proliferation, phosphatidylcholine-phospholipase D may transduce, via phosphatidic acid, negative immunomodulatory signals in mature B lymphocytes.     

Proliferation induced by keratinocyte growth factor enhances in vivo retroviral-mediated gene transfer to mouse hepatocytes

Retroviral gene transfer to liver without prior injury has not yet been accomplished. We hypothesized that recombinant human keratinocyte growth factor would stimulate proliferation of hepatocytes and allow for efficient in vivo gene transfer with high titer murine Moloney retroviral vectors. This report shows that 48 h after intravenous injection of keratinocyte growth factor, hepatocyte proliferation increased approximately 40-fold compared to non-stimulated livers. When keratinocyte growth factor treatment was followed by intravenous injection of high titer (1 x 10(8) colony forming units/ml) retrovirus coding for the Escherichia Coli beta-galactosidase gene, there was a 600-fold increase in beta-galactosidase expression, with 2% of hepatocytes transduced. Thus, by exploiting the mitogenic properties of keratinocyte growth factor, retrovirus-mediated gene transfer to liver may be accomplished in vivo without the use of partial hepatectomy or pretreatment with other toxins to induce hepatocyte cell division.     

The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts

Sequence-dependent formation and lack of repair of polycyclic aromatic hydrocarbon-induced DNA adducts correlates well with the positions of p53 mutational hotspots in smoking-related lung cancers (Denissenko et al, 1996, 1998). The mycotoxin aflatoxin B1 (AFB1) is considered to be a major causative agent in hepatocellular carcinoma (HCC) in regions with presumed high food contamination by AFB1. A unique mutational hotspot, a G to T transversion at the third base of codon 249 of the p53 gene is observed in these tumors. To test whether a selectivity of AFB1 adduct formation is related to this peculiar mutational spectrum, we have mapped AFB1-DNA adducts at nucleotide resolution using ligation-mediated PCR and terminal transferase-dependent PCR. Human HepG2 cells were exposed to AFB1 metabolically activated in the presence of rat liver microsomes. Significant adduct formation was seen at the third base of codon 249. However, this was not the major site of AFB1 adducts and strong adduction was also observed at codons 226, 243, 244, 245 and 248 in exon 7 of the p53 gene and at several codons in exon 8. The damage at codon 249 does not consist of a unique abasic site or ring-opened aflatoxin B1 adduct but rather is consistent with the principal N7-guanine adduct of AFB1. Time course experiments indicate that, under the conditions used, AFB1 adducts are not removed in a strand-selective manner and adduct removal from the third base of codon 249 proceeds at a relatively fast rate (50% in 7 h). The incomplete correspondence between sites of persistent AFB1 damage and the specific codon 249 mutation suggests that AFB1 may not be involved in mutation of this site or that additional mechanisms such as parallel infection with hepatitis B virus may be required for selection of codon 249 mutants in HCC.     

Amyloid fibres of Sup35 support a prion-like mechanism of inheritance in yeast

Hydrogels were prepared from poly(vinyl alcohol) and chitosan in various blend ratios. The water contents of the hydrogels were in the range of 65 to 75 wt %. The attachment and growth of fibroblast cells (L-929) on the hydrogels were studied with a cell culture method. On the hydrogels with more than 15 wt % chitosan content, the attached cells were able not only to remain viable but also to proliferate. The relative cell attachment after incubation for 30 h increased with increasing chitosan content in the hydrogels. Cell attachment and growth on the hydrogel with 40 wt % chitosan content exceeded those on collagen, a widely-used mammalian cell culture substrate. The morphology of the cells attached onto the hydrogels with a lower chitosan content was spherical, but in hydrogels with more than 15 wt % chitosan content, the number of spindle-shaped cells increased with increasing chitosan content.     

Three-dimensional echocardiographic evaluation of fetal heart anatomy and function: acquisition, analysis, and display

The purpose of this work was to assess the functional dynamics and anatomy of the cardiac chambers and great vessels in the fetus (18 to 36 weeks) using in utero three-dimensional ultrasonographic imaging. Fifteen patients were studied using conventional two-dimensional sonographic equipment incorporating a position sensor attached to the transducer and a graphics workstation. Sonographic image data were acquired at 30 images per second and required less than 30 seconds per data set. Fetal heart rate and time in the cardiac cycle were determined and used to synchronize image data for reprojection into a volume at the appropriate part of the cardiac cycle. Volume data were analyzed, rendered, and displayed interactively. Three-dimensional sonographic volume data demonstrated fetal cardiac anatomy from multiple orientations and showed the myocardium, valves, ventricles, and atria clearly. The images showed good correlation with currently available embryologic-anatomic-pathologic data. Dynamic and spatial relationships among chambers, valves, and great vessels were readily appreciated. Three-dimensional sonographic imaging of the fetal heart provides both anatomic and functional information regarding the valves, myocardium, great vessels, and chamber dynamics. Interactive three-dimensional cinegraphic display enhances visualization of cardiac anatomy, which can be difficult to appreciate with two-dimensional methods. The methods presented in this work demonstrate the feasibility of three-dimensional fetal echocardiography.     

A simple but precise method for quantitative measurement of the quality of the laser focus in a scanning optical microscope

We report a method for characterizing the focussing laser beam exiting the objective in a laser scanning microscope. This method provides the size of the optical focus, the divergence of the beam, the ellipticity and the astigmatism. We use a microscopic‐scale knife edge in the form of a simple transmission electron microscopy grid attached to a glass microscope slide, and a light‐collecting optical fibre and photodiode underneath the specimen. By scanning the laser spot from a reflective to a transmitting part of the grid, a beam profile in the form of an error function can be obtained and by repeating this with the knife edge at different axial positions relative to the beam waist, the divergence and astigmatism of the postobjective laser beam can be obtained. The measured divergence can be used to quantify how much of the full numerical aperture of the lens is used in practice. We present data of the beam radius, beam divergence, ellipticity and astigmatism obtained with low (0.15, 0.7) and high (1.3) numerical aperture lenses and lasers commonly used in confocal and multiphoton laser scanning microscopy. Our knife‐edge method has several advantages over alternative knife‐edge methods used in microscopy including that the knife edge is easy to prepare, that the beam can be characterized also directly under a cover slip, as necessary to reduce spherical aberrations for objectives designed to be used with a cover slip, and it is suitable for use with commercial laser scanning microscopes where access to the laser beam can be limited.     

Virus–Host Interaction Gets Curiouser and Curiouser. PART II: Functional Transcriptomics of the E. coli DksA-Deficient Cell upon Phage P1vir Infection

The virus–host interaction requires a complex interplay between the phage strategy of reprogramming the host machinery to produce and release progeny virions, and the host defense against infection. Using RNA sequencing, we investigated the phage–host interaction to resolve the phenomenon of improved lytic development of P1vir phage in a DksA-deficient E. coli host. Expression of the ant1 and kilA P1vir genes in the wild-type host was the highest among all and most probably leads to phage virulence. Interestingly, in a DksA-deficient host, P1vir genes encoding lysozyme and holin are downregulated, while antiholins are upregulated. Gene expression of RepA, a protein necessary for replication initiating at the phage oriR region, is increased in the dksA mutant; this is also true for phage genes responsible for viral morphogenesis and architecture. Still, it seems that P1vir is taking control of the bacterial protein, sugar, and lipid metabolism in both, the wild type and dksA⁻ hosts. Generally, bacterial hosts are reacting by activating their SOS response or upregulating the heat shock proteins. However, only DksA-deficient cells upregulate their sulfur metabolism and downregulate proteolysis upon P1vir infection. We conclude that P1vir development is enhanced in the dksA mutant due to several improvements, including replication and virion assembly, as well as a less efficient lysis.     

Molecular and functional remodeling of electrogenic membrane of hypothalamic neurons in response to changes in their input

Neurons respond to stimuli by integrating generator and synaptic potentials and generating action potentials. However, whether the underlying electrogenic machinery within neurons itself changes, in response to alterations in input, is not known. To determine whether there are changes in Na+ channel expression and function within neurons in response to altered input, we exposed magnocellular neurosecretory cells (MNCs) in the rat supraoptic nucleus to different osmotic milieus by salt-loading and studied Na+ channel mRNA and protein, and Na+ currents, in these cells. In situ hybridization demonstrated significantly increased mRNA levels for alpha-II, Na6, beta1 and beta2 Na+ channel subunits, and immunohistochemistry/immunoblotting showed increased Na+ channel protein after salt-loading. Using patch-clamp recordings to examine the deployment of functional Na+ channels in the membranes of MNCs, we observed an increase in the amplitude of the transient Na+ current after salt-loading and an even greater increase in amplitude and density of the persistent Na+ current evoked at subthreshold potentials by slow ramp depolarizations. These results demonstrate that MNCs respond to salt-loading by selectively synthesizing additional, functional Na+ channel subtypes whose deployment in the membrane changes its electrogenic properties. Thus, neurons may respond to changes in their input not only by producing different patterns of electrical activity, but also by remodeling the electrogenic machinery that underlies this activity.     

Analysis of annealed thin polymer films prepared from dichloro(methyl)phenylsilane by plasma polymerization

Thin plasma polymer films were deposited from a mixture of dichloro(methyl)phenylsilane (DCMPS) vapor and gaseous hydrogen in an rf (13.56 MHz) capacitive coupling deposition system on pieces of silicon wafers. Some of samples were annealed in a vacuum to temperatures ranging from 450 to 700°C. The chemical composition, structure, and surface morphology of the annealed samples and those stored in air at room temperature were studied by FTIR, XPS, SEM, and optical microscopy. The thermal stability and decomposition of the plasma polymer with increasing temperature were characterized using thermogravimetry together with mass spectrometry. The plasma polymer was stable to a temperature of 300°C. Above that temperature, the material started to decompose together with additional crosslinking due to the incorporation of extra oxygen atoms forming new siloxane bonds. The plasma polymer was tough at room temperature but much more brittle after annealing. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 82: 2106–2112, 2001