The effect of pre-operative blood withdrawal, with or without sequestration, on allogeneic blood product requirements.

UNLABELLED: A common effect of autologous blood withdrawal before cardiopulmonary bypass (CPB) is a decrease in haematocrit (Hct) and haemoglobin (Hb) content. A refinement of this technique is autologous blood withdrawal with the sequestration of platelet rich plasma (PRP) and red blood cells (RBCs). METHODS: One hundred and four patients were included in a randomized study stratified into three groups: the autologous blood withdrawal group (Group 1), the autologous blood withdrawal group with blood loss sequestration (Group 2) and the control group (Control group). In Group 1, the amount of withdrawn blood was transfused after CPB. In Group 2, the RBCs were transfused immediately after sequestration and the PRP was transfused after the termination of CPB. In the Control group, no autologous blood withdrawal was employed. The following variables were analysed: blood loss, blood products transfusion, fluid transfusion, diuresis, haematological and coagulation data and the duration of the operation and intensive care unit stay. RESULTS: We found no significant differences in peri-operative blood loss and transfused blood products among the three groups. There was a trend towards a lower amount of transfused fresh frozen plasma (FFP) for Group 1 (p =0.057) in the operation room (OR). The use of plasma expanders post-CPB was significantly higher in the Control group (p=0.024). RBCs coming from the auto-transfusion device were, for Group 1, significantly lower (p=0.007). The Hb and Hct values in Group 1, at start and end of CPB, were significantly lower (p=0.023-0.003 / 0.001-0.001, respectively). All other parameters were not significantly different. CONCLUSION: there were no significant differences between the study groups. This randomized trial shows that, although sequestration immediately after autologous blood withdrawal has no added value, autologous blood withdrawal in patients with a normal pre-operative Hb and Hct is simple, inexpensive and allows for autologous blood transfusion.

Endstation for ultrafast magnetic scattering experiments at the free-electron laser in Hamburg.

An endstation for pump-probe small-angle X-ray scattering (SAXS) experiments at the free-electron laser in Hamburg (FLASH) is presented. The endstation houses a solid-state absorber, optical incoupling for pump-probe experiments, time zero measurement, sample chamber, and detection unit. It can be used at all FLASH beamlines in the whole photon energy range offered by FLASH. The capabilities of the setup are demonstrated by showing the results of resonant magnetic SAXS measurements on cobalt-platinum multilayer samples grown on freestanding Si(3)N(4) membranes and pump-laser-induced grid structures in multilayer samples.

Breakdown of the x-ray resonant magnetic scattering signal during intense pulses of extreme ultraviolet free-electron-laser radiation.

We present results of single-shot resonant magnetic scattering experiments of Co/Pt multilayer systems using 100 fs long ultraintense pulses from an extreme ultraviolet (XUV) free-electron laser. An x-ray-induced breakdown of the resonant magnetic scattering channel during the pulse duration is observed at fluences of 5 J/cm(2). Simultaneously, the speckle contrast of the high-fluence scattering pattern is significantly reduced. We performed simulations of the nonequilibrium evolution of the Co/Pt multilayer system during the XUV pulse duration. We find that the electronic state of the sample is strongly perturbed during the first few femtoseconds of exposure leading to an ultrafast quenching of the resonant magnetic scattering mechanism.

Ultrafast optical demagnetization manipulates nanoscale spin structure in domain walls.

During ultrafast demagnetization of a magnetically ordered solid, angular momentum has to be transferred between the spins, electrons, and phonons in the system on femto- and picosecond timescales. Although the intrinsic spin-transfer mechanisms are intensely debated, additional extrinsic mechanisms arising due to nanoscale heterogeneity have only recently entered the discussion. Here we use femtosecond X-ray pulses from a free-electron laser to study thin film samples with magnetic domain patterns. We observe an infrared-pump-induced change of the spin structure within the domain walls on the sub-picosecond timescale. This domain-topography-dependent contribution connects the intrinsic demagnetization process in each domain with spin-transport processes across the domain walls, demonstrating the importance of spin-dependent electron transport between differently magnetized regions as an ultrafast demagnetization channel. This pathway exists independent from structural inhomogeneities such as chemical interfaces, and gives rise to an ultrafast spatially varying response to optical pump pulses.

Photodynamic therapy mediated induction of accelerated re-endothelialisation following injury to the arterial wall: implications for the prevention of postinterventional restenosis.

OBJECTIVE: Accelerated re-endothelialisation may inhibit the development of restenosis. Basic Fibroblast Growth Factor (bFGF) plays a key role for early proliferative activity in the artery following injury. Therefore, this study was devised to examine the effect of photodynamic therapy (PDT) on post-injury re-endothelialisation in vivo, and bFGF-mRNA expression in endothelial cells (EC) in vitro. MATERIALS AND METHODS: Rat carotid arteries were balloon-injured prior to PDT. Arteries were analysed after 1, 3, 5, 14 and 30 days. Morphometric measurements were undertaken following injection of 0.5% Evans Blue which stains non-endothelialised surfaces only. To identify EC, immunohistochemistry (CD-31) was performed. Proliferation was assessed by fluorescence cell counting. PCR quantification of bFGF-mRNA expression and proliferation were assessed in bovine aortic EC which were plated on isolated, PDT-treated EC-derived extracellular matrix at (12), 24, 48 (72 h). RESULTS: Three days following PDT, arteries displayed significantly increased endothelial lining (p = 0.02), which was more pronounced at 5 (p = 0.03) and 14 days (p = 0.02). At 30 days no relevant differences between PDT and control were noted. EC proliferation on PDT-treated matrix was significantly increased at 24, 48, and 72 h (p = 0.0004), whereas bFGF-mRNA expression was significantly increased at 24 h only (p = 0.007). CONCLUSION: Post-injury PDT appears to accelerate re-endothelialisation. Expression of bFGF-mRNA, however, although increased shortly after PDT, may not be responsible for a constant stimulation of EC proliferation.

Dual role of GdmH in producer immunity and secretion of the Staphylococcal lantibiotics gallidermin and epidermin.

The biosynthetic gene clusters of the staphylococcal lantibiotics epidermin and gallidermin are distinguished by the presence of the unique genes epiH and gdmH, respectively. They encode accessory factors for the ATP-binding cassette transporters that mediate secretion of the antimicrobial peptides. Here, we show that gdmH also contributes to immunity to gallidermin but not to nisin. gdmH alone affected susceptibility to gallidermin only moderately, but it led to a multiplication of the immunity level mediated by the FEG immunity genes when cloned together with the gdmT gene, suggesting a synergistic activity of the H and FEG systems. gdmH-related genes were identified in the genomes of several bacteria, indicating an involvement in further cellular functions.

Roles for zebrafish focal adhesion kinase in notochord and somite morphogenesis.

We have cloned zebrafish focal adhesion kinase (Fak) and analyzed its subcellular localization. Fak protein is localized at the cortex of notochord cells and at the notochord-somite boundary. During somitogenesis, Fak protein becomes concentrated at the basal region of epithelial cells at intersomitic boundaries. Phosphorylated Fak protein is seen at both the notochord-somite boundary and intersomitic boundaries, consistent with a role for Fak in boundary formation and maintenance. The localization of Fak protein to the basal region of epithelial cells in knypek;trilobite double mutant embryos shows that polarization of Fak distribution in the somite border cells is independent of internal mesenchymal cells. In addition, we show that neither Notch signaling through Suppressor of Hairless (SuH) nor deltaD is necessary for the wild-type segmental pattern of fak mRNA expression in the anterior paraxial mesoderm. However, nonsegmental expression of fak mRNA occurs with ectopic activation of Notch signaling through SuH and also in fused somite and beamter mutant embryos, indicating that there are multiple regulators of fak mRNA expression. Our results suggest that Fak plays a central role in notochord and somite morphogenesis.

Protein kinase C-related kinase 2 phosphorylates the protein synthesis initiation factor eIF4E in starfish oocytes.

Phosphorylation of eIF4E is required for protein synthesis during starfish oocyte maturation. The activity of protein kinase C-related kinase 2 (PRK2) increases prior to the phosphorylation of eIF4E (G. Stapleton et al., 1998, Dev. Biol. 193, 34-46). We investigate here whether eIF4E is activated by PRK2. A 3.5-kb eIF4E clone isolated from starfish cDNA is 57% identical to human eIF4E and contains the putative phosphorylation site serine-209. The serine-209 environment (SKTGS(209)MAKSRF) is similar to the consensus sequence of the phosphorylation site of protein kinase C and related kinases. A starfish eIF4E fusion protein (GST-4E) was phosphorylated in vitro by PRK2 in the presence of 1,2-diolyl-sn-glycerol 3-phosphate. In contrast, replacing the GST-4E serine-209 with an alanine significantly reduced this phosphorylation. Analysis by two-dimensional phosphopeptide mapping reveals a major phosphopeptide in trypsin-digested GST-4E, but not in its serine-209 mutant. Importantly, this major phosphopeptide in GST-4E corresponds to a major phosphopeptide of eIF4E isolated from (32)P-labeled oocytes. Thus, PRK2 may regulate translation initiation during oocyte maturation by phosphorylating the serine-209 residue of eIF4E in starfish. We also demonstrate that high levels of cAMP inhibit the activation of PRK2, eIF4E, and the eIF4E binding protein during starfish oocyte maturation, while PI3 kinase activates these proteins.

Somites in zebrafish doubly mutant for knypek and trilobite form without internal mesenchymal cells or compaction.

In vertebrates, paraxial mesoderm is partitioned into repeating units called somites. It is thought that the mechanical forces arising from compaction of the presumptive internal cells of prospective somites cause them to detach from the unsegmented presomitic mesoderm [1-3]. To determine how prospective somites physically segregate from each other, we used time-lapse microscopy to analyze the mechanics underlying early somitogenesis in wild-type zebrafish and in the mutants trilobite(m209) (tri), knypek(m119) (kny), and kny;tri, which are defective in convergent extension during gastrulation. Formation of somite boundaries in all of these embryos involved segregation, local alignment, and cell-shape changes of presumptive epitheloid border cells along nascent intersomitic boundaries. Although kny;tri somites formed without convergence of the presomitic mesoderm and were composed of only two cells in their anteroposterior (AP) dimension, they still exhibited AP intrasegmental polarity. Furthermore, morphogenesis of somite boundaries in these embryos proceeded in a manner similar to that in wild-type embryos. Thus, intersomitic boundary formation in zebrafish involves short-range movements of presumptive border cells that do not require mechanical forces generated by internal cells or compaction of the presomitic mesoderm.

Phosphorylation of protein kinase C-related kinase PRK2 during meiotic maturation of starfish oocytes.

The resumption of meiosis in the developing starfish oocyte is the result of intracellular signaling events initiated by 1-methyladenine stimulation. One of the earliest detectable kinase activities during meiotic maturation of starfish oocytes is a protein kinase C or PKC-like activity. In this study, several isoforms of protein kinase C were cloned from the oocyte; however, the most abundant PKC-like maternal transcript corresponds to protein kinase C-related kinase 2 (PRK2). PRK2 is expressed in the immature oocyte and at least until germinal vesicle breakdown. Subcellular localization of PRK2 revealed a cytoplasmic distribution in the immature oocyte, which, during meiotic maturation, remained in the cytoplasm but also localized to the disintegrating germinal vesicle. Significantly, PRK2 is phosphorylated in vivo in response to 1-methyladenine which precedes MPF activation, making PRK2 a candidate regulator of early signaling events of meiotic maturation.

Secretion of the lantibiotics epidermin and gallidermin: sequence analysis of the genes gdmT and gdmH, their influence on epidermin production and their regulation by EpiQ.

The closely related lantibiotics epidermin and gallidermin are produced by Staphylococcus epidermidis Tu3298 and S. gallinarum Tu3928, respectively. The epidermin biosynthetic genes involved in maturation, regulation, and immunity have been identified previously. How epidermin or gallidermin is secreted, however, has remained unclear. Here, we characterize two additional genes, epiH and epiT, as well as the homologous gallidermin genes gdmH and gdmT. EpiT and GdmT are similar to one-component ABC transporters that are involved in the secretion of proteins or peptides. EpiH and GdmH are hydrophobic proteins without conspicuous similarities to other proteins. Comparison of the gene sequences revealed that epiT is incomplete, having an internal deletion that causes a frame shift and a second deletion at the 3'-end, while gdmT is intact. Introduction of epiT and epiH into the heterologous host S. carnosus (pTepi14) bearing the maturation and regulation genes had no significant effect on the rather low level of epidermin production. The presence of the homologous gdmT and gdmH, however, resulted in a strong increase (seven- to tenfold) in the production level, which is very likely to be due to increased efficiency of epidermin secretion. Both gdmT and gdmH were necessary for this effect, indicating that the two gene products cooperate in some way. In the epidermin-producing wild-type strain Tu3298, which contains epiH and the disrupted epiT, the addition of gdmT alone led to a two-fold increase in epidermin production. Both gdmT and gdmH and the corresponding epi genes were activated by the transcriptional regulator EpiQ; this is in accordance with the presence of putative EpiQ operator sites in the promoter regions.

Modulation of the inhibitory substrate properties of oligodendrocytes by platelet-derived growth factor.

Although growth cones typically collapse after encountering O1/galactocerebroside (GalC)-positive oligodendrocytes, the majority of growth cones traversed oligodendrocytes, which were raised for 8-10 d in medium containing 10 ng/ml platelet-derived growth factor (PDGF). Oligodendrocytes raised 8-10 d in control medium caused growth cone collapse as they normally do, but failed to elicit this response after being transferred to PDGF-containing medium for an additional 8-10 d. The opposite was observed when PDGF-treated oligodendrocytes were brought to control medium. Growth cones collapsed when contacting these cells. Oligodendrocytes also lost their collapse-inducing activity when raised in medium conditioned by astrocytes, known to produce PDGF. Antibody IN-1 is directed against against neurite growth inhibitors (NI), proteins of 35 and 250 kDa on the surface of O1/GalC-positive oligodendrocytes, which are known to elicit growth cone collapse. IN-1 immunoreactivity was markedly reduced in PDGF-treated oligodendrocytes. However, both PDGF-treated and control oligodendrocytes exhibited myelin-associated glycoprotein, proteolipid protein, and myelin basic protein immunoreactivity. This suggests that PDGF-treatment affects NI expression but does not interfere with the expression of advanced myelin marker proteins. Because NI cause growth cone collapse, the loss of collapse-inducing activity by PDGF-treated oligodendrocytes suggests that PDGF regulates, directly or indirectly, the expression of these proteins.

A dominant inhibitory mutant of the type II transforming growth factor beta receptor in the malignant progression of a cutaneous T-cell lymphoma.

In many cancers, inactivating mutations in both alleles of the transforming growth factor beta (TGF-beta) type 11 receptor (TbetaRII) gene occur and correlate with loss of sensitivity to TGF-beta. Here we describe a novel mechanism for loss of sensitivity to growth inhibition by TGF-beta in tumor development. Mac-1 cells, isolated from the blood of a patient with an indolent form of cutaneous T-cell lymphoma, express wild-type TbetaRII and are sensitive to TGF-beta. Mac-2A cells, clonally related to Mac-1 and isolated from a skin nodule of the same patient at a later, clinically aggressive stage of lymphoma, are resistant to TGF-beta. They express both the wild-type TbetaRII and a receptor with a single point mutation (Asp-404-Gly [D404G]) in the kinase domain (D404G-->TbetaRII); no TbetaRI or TbetaRII is found on the plasma membrane, suggesting that D404G-TbetaRII dominantly inhibits the function of the wild-type receptor by inhibiting its appearance on the plasma membrane. Indeed, inducible expression, under control of a tetracycline-regulated promoter, of D404G-TbetaRII in TGF-beta- sensitive Mac-1 cells as well as in Hep3B hepatoma cells results in resistance to TGF-beta and disappearance of cell surface TbetaRI and TbetaRII. Overexpression of wild-type TbetaRII in Mac-2A cells restores cell surface TbetaRI and TbetaRH and sensitivity to TGF-beta. The ability of the D404G-TbetaRH to dominantly inhibit function of wild-type TGF-beta receptors represents a new mechanism for loss of sensitivity to the growth-inhibitory functions of TGF-beta in tumor development.

Gastrulation in the sea urchin, Strongylocentrotus purpuratus, is disrupted by the small laminin peptides YIGSR and IKVAV.

Laminin is present on the apical and basolateral sides of epithelial cells of very early sea urchin blastulae. We investigated whether small laminin-peptides, known to have cell binding activities, alter the development of sea urchin embryos. The peptide YIGSR-NH2 (850 microM) and the peptide PA22-2 (5 microM), which contains the peptide sequence IKVAV (Tashiro et al., J. Biol. Chem. 264, 16174, 1989), typically blocked archenteron formation when added to the sea water soon after fertilization. At lower doses, the YIGSR peptide allowed invagination of the archenteron but blocked archenteron extension and differentiation and evagination of the feeding arms. The effect of YIGSR and PA22-2 peptides declined when added to progressively older stages until no effect was seen when added at the mesenchyme blastula stage (24 hours after fertilization). Control peptides GRGDS, YIGSE, and SHA22, a dodeca-peptide with a scrambled IKVAV sequence, had no effect on development. The YIGSK peptide containing a conserved amino acid modification had only a small effect on gastrulation. The results suggest that YIGSR and IKVAV peptides specifically disrupt cell/extracellular matrix interactions required for normal development of the archenteron and feeding arms. Our recent finding that YTGIR is at the cell binding site of the B1 chain of S. purpuratus laminin supports this conclusion. Evidently, laminin or other laminin-like molecules are among the many extracellular matrix components needed for the invagination and extension of the archenteron during the gastrulation movements of these embryos.

Maturation hormone induced an increase in the translational activity of starfish oocytes coincident with the phosphorylation of the mRNA cap binding protein, eIF-4E, and the activation of several kinases.

The stimulation of translation in starfish oocytes by the maturation hormone, 1-methyladenine (1-MA), requires the activation or mobilization of both initiation factors and mRNAs [Xu and Hille, Cell Regul. 1:1057, 1990]. We identify here the translational initiation complex, eIF-4F, and the guanine nucleotide exchange factor for eIF-2, eIF-2B, as the rate controlling components of protein synthesis in immature oocytes of the starfish, Pisaster orchraceus. Increased phosphorylation of eIF-4E, the cap binding subunit of the eIF-4F complex, is coincident with the initial increase in translational activity during maturation of these oocytes. Significantly, protein kinase C activity increased during oocyte maturation in parallel with the increase in eIF-4E phosphorylation and protein synthesis. An increase in the activities of cdc2 kinase and mitogen-activated myelin basic protein kinase (MBP kinase) similarly coincide with the increase in eIF-4E phosphorylation. However, neither cdc2 kinase nor MBP kinase phosphorylates eIF-4E in vitro. Casein kinase II activity does not change during oocyte maturation, and therefore, cannot be responsible for the activation of translation. Treatment of oocytes with phorbol 12-myristate 13-acetate, an activator of protein kinase C, for 30 min prior to the addition of 1-MA resulted in the inhibition of 1-MA-induced phosphorylation of eIF-4E, translational activation, and germinal vesicle breakdown. Therefore, protein kinase C may phosphorylate eIF-4E, after very early events of maturation. Another possibility is that eIF-4E is phosphorylated by an unknown kinase that is activated by the cascade of reactions stimulated by 1-MA. In conclusion, our results suggest a role for the phosphorylation of eIF-4E in the activation of translation during maturation, similar to translational regulation during the stimulation of growth in mammalian cells.

Clinical features of vascular thrombosis following varicella.

OBJECTIVE: To define the clinical characteristics, neuroimaging features, and outcome of five patients with post-primary varicella zoster virus infection hemiparesis and to offer a hypothesis to explain the predilection for the involvement of the cerebral vasculature in this condition. DESIGN: Patient series. SETTING: Five patients were treated during a 14-month period in a private pediatric neurology practice in a medium-size southwestern city. INTERVENTIONS: Steroids (two patients) and antiplatelet drugs (two patients). No observed effects of therapy. RESULTS: The onset of the hemiparesis occurred several weeks (mean, 5.4 weeks) following an episode of the chickenpox. Magnetic resonance imaging was more sensitive than computed tomography or angiography in demonstrating the area of involvement deep in the cerebral hemispheres. The prognosis was good regardless of the therapy administered, as all patients recovered completely or nearly completely. CONCLUSIONS: Primary varicella zoster virus infection with delayed-onset hemiparesis typically occurs approximately 6 weeks after primary varicella zoster virus infection. Magnetic resonance imaging is the most sensitive neuroimaging tool in these children. The prognosis is good, with recovery of function and no recurrences in our patients. The innervation of the carotid artery and the characteristics of the varicella zoster virus itself together provide the local and systemic factors that may trigger the vasculopathy responsible for this syndrome.

Cell-free translation systems prepared from starfish oocytes faithfully reflect in vivo activity; mRNA and initiation factors stimulate supernatants from immature oocytes.

Meiotic maturation stimulates a change in the translation of stored mRNAs: mRNAs encoding proteins needed for growth of oocytes are translated before meiotic maturation, whereas those encoding proteins required for cleavage are translated after meiotic maturation. Studies of translational regulation during meiotic maturation have been limited by the lack of translationally active cell-free supernatants. Starfish oocytes are ideal for preparing cell-free translation systems because experimental application of the hormone 1-methyladenine induces their maturation, synchronizing meiosis. We have prepared such systems from both immature and mature oocytes of starfish. Changes in protein synthesis rates and the specificity of proteins synthesized in these cell-free translation supernatants mimic those seen in vivo. Supernatants both from immature and mature oocytes have a high capacity to initiate new translation because 90% of the proteins made are newly initiated from mRNAs. Cell-free supernatants from mature oocytes have a much higher rate of initiation of translation than those from immature oocytes and use the 43S preinitiation complexes more efficiently in initiation of translation. Similarly, we have shown that mRNAs and initiation factors are rate limiting in cell-free translation systems prepared from immature oocytes. In addition, cell-free translation systems prepared from immature oocytes are only slightly, if at all, inhibitory to cell-free translation systems from mature oocytes. Thus, soluble inhibitors, if they exist, are rapidly converted by cell-free supernatants from mature oocytes. The similarities between translation in our starfish cell-free translation systems and in intact oocytes suggests that the cell-free translation systems will be useful tools for further studies of maturation events and translational control during meiosis.

Purification and characterization of sea urchin initiation factor 2. The requirement of guanine nucleotide exchange factor for the release of eukaryotic polypeptide chain initiation factor 2-bound GDP.

Protein synthesis in sea urchin eggs is stimulated dramatically upon fertilization. We previously demonstrated that this stimulation is primarily due to an increase in the rate of polypeptide chain initiation which in turn may be regulated at the level of recycling of eukaryotic initiation factor 2 (eIF-2) (Colin, A. M., Brown, B. D., Dholakia, J. N., Woodley, C. L., Wahba, A. J., and Hille, M. B. (1987) Dev. Biol. 123, 354-363). We have now purified eIF-2 from sea urchin Strongylocentrotus purpuratus blastulae to apparent homogeneity by chromatography on DEAE-cellulose, phosphocellulose, Mono Q, Mono P, and Mono S columns. The factor, which differs from mammalian eIF-2, is composed of three non-identical subunits with apparent molecular weights of 40,000-alpha; 47,000-beta, and 58,000-gamma as estimated by sodium dodecyl-polyacrylamide gel electrophoresis. Antibodies raised against rabbit reticulocyte eIF-2 do not cross-react with sea urchin eIF-2. The binding of Met-tRNA(f) to sea urchin eIF-2 is totally dependent on GTP. A 4-fold stimulation in the rate of protein synthesis in unfertilized sea urchin egg extracts is observed by the addition of 1 micrograms of purified eIF-2. The factor also binds GDP to form a binary (eIF-2.GDP) complex which is stable in the presence of Mg2+. GDP binding to sea urchin eIF-2 inhibits ternary (eIF-2-GTP.[35S]Met-tRNA(f) complex formation. The rabbit reticulocyte guanine nucleotide exchange factor (GEF) catalyzes the exchange of GDP bound to sea urchin eIF-2 for GTP and stimulates ternary complex formation. The requirement of GEF for the recycling of eIF-2 suggests that protein synthesis in sea urchins is similar to that in mammalian systems and may also be regulated at the level of GEF activity. The reticulocyte heme-controlled repressor phosphorylates the alpha-subunit of eIF-2 from both sea urchins and rabbit reticulocytes. However, casein kinase II which phosphorylates the beta-subunit of the reticulocyte factor specifically phosphorylates the alpha-subunit of sea urchin eIF-2. In this respect, the sea urchin factor is similar to eIF-2 isolated from other nonmammalian sources. Since both heme controlled repressor and casein kinase II phosphorylate the alpha-subunit of sea urchin eIF-2 caution should be exercised when interpreting the significance of eIF-2(alpha) phosphorylation in sea urchins.