The effects and comparative differences of neutrophil specific chemokines on neutrophil chemotaxis of the neonate

Neutrophil specific chemokines are potent chemoattractants for neutrophils. IL-8/CXCL8 is the most extensively studied member of this group, and its concentrations increase during inflammatory conditions of the newborn infant including sepsis and chronic lung disease. A significant amount of information exists on the effects of IL-8/CXCL8 on neutrophil chemotaxis of neonates, but little is known about the other neutrophil specific chemokines. The aim of this study was to determine the relative potency of the neutrophil specific chemokines on chemotaxis of neonatal neutrophils and to compare this effect with the effect on adult neutrophils. Neutrophils were isolated from cord blood or healthy adult donors and incubated in a Neuroprobe chemotaxis chamber. Chemokine concentrations ranging from 1-1000 ng/mL were used. Differences in chemotactic potency existed among the seven neutrophil specific chemokines. Specifically, at 100 ng/mL, the order was IL-8/CXCL8>GRO-alpha/CXCL1>GCP-2/CXCL6>NAP-2/CXCL7>ENA-78/CXCL5>GRO-gamma/CXCL2>GRO-beta/CXCL3. This pattern was observed for adult and neonatal neutrophils. We conclude that (1) neutrophils from cord blood exhibit the same pattern of potency for each ELR chemokine as neutrophils from adults, and (2) migration of neonatal neutrophils is significantly less than that of adults at every concentration examined except the lowest (1 ng/mL).     

Altered gene expression in plants with constitutive expression of a mitochondrial small heat shock protein suggests the involvement of retrograde regulation in the heat stress response

Heat stress can negatively affect crop productivity. One way in which plants attempt to alleviate the effects of heat stress is to induce the expression of genes encoding heat shock proteins (HSPs), including small HSPs (sHSPs). We produced transgenic lines of Arabidopsis thaliana expressing a transgene encoding a maize mitochondrial sHSP, ZmHSP22. The transgene, under the control of the cauliflower mosaic virus 35S promoter, is constitutively highly expressed in these lines. As demonstrated by confocal immunofluorescence microscopy and analyses of isolated mitochondria, ZmHSP22 is directed to the mitochondria of Arabidopsis and is processed into the mature form. These transgenic lines demonstrated altered expression of nuclear genes encoding the endogenous mitochondrial sHSP, AtHSP23.6, chloroplast localized AtHSP25.3, class I cytosolic AtHSP17.4, cytosolic AtHSP70-1 and chloroplast localized AtHSP70-6, but not cytosolic AtHSP70-15, following exposure to heat stress. This suggests that the expression of HSPs can be affected by heat-induced mitochondrial retrograde regulation. Three-week-old plants from the transgenic Arabidopsis lines expressing ZmHSP22 have increased thermotolerance, as measured by the maintenance of higher leaf mass following successive days with short periods of heat stress.     

Studies on marine fungal-nematode associations and plant degradation

Summary 1. Studies of the broad-leafed turtle grass,Thalassia testudinum König, have revealed a diverse range of fungal infestation different in generic composition and dynamics of attack from that found on submerged wood. Certain of the fungi, notably the AscomyceteLindra thalassiae, initiate considerable degradation of leaf tissue and show a developmental cycle in nature related to the physiological state of the host plant.2. Use of fungal-cellulose mats as a trapping substrate has been extremely effective for discernment of ecologically significant shifts in nematode concentrations, especially those of the omnivorous species,Metoncholaimus scissus.3. Patterns of activity ofM. scissus, as well as those of various foliicolous nematodes, suggest that loci of organic material, such as fungal infested leaves and decaying plant tissue, significantly affect biological activity of these animals.4. Laboratory analysis of degraded cotton cellulose filters show a striking incidence of fungal reproduction of the ascomycetous fungusLulworthia, along with development of a considerable associated nematode fauna, especially species ofViscosia (V. macramphida) andLeptolaimus (L. plectoides). Successional patterns in nematode development are noted with continued degradation of the cotton cellulose matrix.

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Studien über marine Pilz-Nematoden-Assoziationen und Pflanzendegradation

Kurzfassung Untersuchungen am SeegrasThalassia testudinum König haben ergeben, daß sich hier Pilzinfektionen hinsichtlich der Komposition der beteiligten Gattungen und der Dynamik des Befalls von den am untergetauchten Holz festgestellten Infektionen unterscheiden. Bestimmte Pilze, insbesondere der AscomycetLindra thalassiae, leiten eine erhebliche Degradation des Blattgewebes ein und zeigen einen Entwicklungszyklus, welcher in Beziehung steht zum physiologischen Zustand der Wirtspflanze. Die Anwendung von Pilz-Zellulose-Matten als Einfangsubstrat war außerordentlich erfolgreich für das Erkennen ökologisch signifikanter Verschiebungen in den Nematodenkonzentrationen, insbesondere bei der omnivoren ArtMetoncholaimus scissus. Die Aktivitätsmuster vonM. scissus — ebenso wie die verschiedener foliicolöser Nematoden — deuten darauf hin, daß pilzinfizierte und zerfallende Pflanzenteile in entscheidendem Maße die biologische Aktivität dieser Tiere beeinflussen. Laboratoriumsanalysen degradierter Wollzellulosefilter lassen eine überraschend starke Vermehrung des AscomycetenLulworthia erkennen und gleichzeitig die Entwicklung einer beachtlichen Fauna assoziierter Nematodenarten, insbesondere vonViscosia macramphida undLeptolaimus plectoides. Im Verlaufe der weiteren Degradation der Wollzellulosematrix kommt es bei der Nematodenfauna zu entsprechenden Sukzessionen.

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Contribution No. 768 from the Institute of Marine Science, University of Miami, Miami, Florida, and from the Canada Department of Agriculture, Ottawa, Canada. This work was supported at the IMS by grant GM 12842 from the National Institutes of Health, USA.     

Ligand Independence of the T618I Mutation in the Colony-stimulating Factor 3 Receptor (CSF3R) Protein Results from Loss of O-Linked Glycosylation and Increased Receptor Dimerization

Mutations in the CSF3 granulocyte colony-stimulating factor receptor CSF3R have recently been found in a large percentage of patients with chronic neutrophilic leukemia and, more rarely, in other types of leukemia. These CSF3R mutations fall into two distinct categories: membrane-proximal mutations and truncation mutations. Although both classes of mutation have exhibited the capacity for cellular transformation, several aspects of this transformation, including the kinetics, the requirement for ligand, and the dysregulation of downstream signaling pathways, have all been shown to be discrepant between the mutation types, suggesting distinct mechanisms of activation. CSF3R truncation mutations induce overexpression and ligand hypersensitivity of the receptor, likely because of the removal of motifs necessary for endocytosis and degradation. In contrast, little is known about the mechanism of activation of membrane-proximal mutations, which are much more commonly observed in chronic neutrophilic leukemia. In contrast with CSF3R truncation mutations, membrane-proximal mutations do not exhibit overexpression and are capable of signaling in the absence of ligand. We show that the Thr-615 and Thr-618 sites of membrane-proximal mutations are part of an O-linked glycosylation cluster. Mutation at these sites prevents O-glycosylation of CSF3R and increases receptor dimerization. This increased dimerization explains the ligand-independent activation of CSF3R membrane-proximal mutations. Cytokine receptor activation through loss of O-glycosylation represents a novel avenue of aberrant signaling. Finally, the combination of the CSF3R membrane proximal and truncation mutations, as has been reported in some patients, leads to enhanced cellular transformation when compared with either mutation alone, underscoring their distinct mechanisms of action.     

Guide wire assisted catheterization and colored dye injection for vascular mapping of monochorionic twin placentas

Monochorionic (MC) twin pregnancies are associated with significantly higher morbidity and mortality rates than dichorionic twins. Approximately 50% of MC twin pregnancies develop complications arising from the shared placenta and associated vascular connections. Severe twin-to-twin syndrome (TTTS) is reported to account for approximately 20% of these complications. Inter-twin vascular connections occur in almost all MC placentas and are related to the prognosis and outcome of these high-risk twin pregnancies. The number, size and type of connections have been implicated in the development of TTTS and other MC twin conditions. Three types of inter-twin vascular connections occur: 1) artery to vein connections (AVs) in which a branch artery carrying deoxygenated blood from one twin courses along the fetal surface of the placenta and dives into a placental cotyledon. Blood flows via a deep intraparenchymal capillary network into a draining vein that emerges at the fetal surface of the placenta and brings oxygenated blood toward the other twin. There is unidirectional flow from the twin supplying the afferent artery toward the twin receiving the efferent vein; 2) artery to artery connections (AAs) in which a branch artery from each twin meets directly on the superficial placental surface resulting in a vessel with pulsatile bidirectional flow, and 3) vein to vein connections (VVs) in which a branch vein from each twin meets directly on the superficial placental surface allowing low pressure bidirectional flow. In utero obstetric sonography with targeted Doppler interrogation has been used to identify the presence of AV and AA connections. Prenatally detected AAs that have been confirmed by postnatal placental injection studies have been shown to be associated with an improved prognosis for both twins. Furthermore, fetoscopic laser ablation of inter-twin vascular connections on the fetal surface of the shared placenta is now the preferred treatment for early, severe TTTS. Postnatal placental injection studies provide a valuable method to confirm the accuracy of prenatal Doppler ultrasound findings and the efficacy of fetal laser therapy. Using colored dyes separately hand-injected into the arterial and venous circulations of each twin, the technique highlights and delineates AVs, AAs, and VVs. This definitive demonstration of MC placental vascular anatomy may then be correlated with Doppler ultrasound findings and neonatal outcome to enhance our understanding of the pathophysiology of MC twinning and its sequelae. Here we demonstrate our placental injection technique.     

The mechanical significance of morphological variation in the macaque mandibular symphysis during mastication

Catarrhine symphyseal morphology displays considerable variation. Although this has been related to dentition, phylogeny, sexual dimorphism, and facial orientation, most emphasis has been given to the functional significance of the symphysis to mechanical loading during mastication. The current state of knowledge regarding the mechanical significance of the symphysis is based on a combination of in vivo experimental and comparative studies on Macaca fascicularis. These approaches have provided considerable insight into the stereotypical patterns of loading in the symphyseal region during chewing and hypotheses related to the associated symphyseal morphologies. Finite element analysis (FEA) was used to assess how in silico manipulation translates into the mechanical loading hypotheses previously proposed experimentally. In particular, this study tests the form-function relationship of the symphysis of an adult M. fascicularis mandible during lateral transverse bending and dorsoventral shear of the mandibular symphysis, and a series of modified hypothetical morphologies including absence/presence of tori and variation in the inclination and depth of the symphysis. FEA results of this study support previous findings that stresses associated with lateral transverse bending and dorsoventral shear of the mandibular symphysis can be minimized via an increased labio-lingual thickness in the superior transverse torus, an oblique symphyseal inclination, and/or an increased symphyseal depth. The finding that reduction of strains related to lateral transverse bending and dorsoventral shear can be achieved through a number of different morphologies contributes to our understanding of the influence of morphological and/or developmental constraints, such as dental development, on symphyseal form.     

Quantitative phosphoproteome analysis of lysophosphatidic acid induced chemotaxis applying dual-step (18)O labeling coupled with immobilized metal-ion affinity chromatography

Reversible protein phosphorylation is a central cellular regulatory mechanism in modulating protein activity and propagating signals within cellular pathways and networks. Development of more effective methods for the simultaneous identification of phosphorylation sites and quantification of temporal changes in protein phosphorylation could provide important insights into molecular signaling mechanisms in various cellular processes. Here we present an integrated quantitative phosphoproteomics approach and its application for comparative analysis of Cos-7 cells in response to lysophosphatidic acid (LPA) gradient stimulation. The approach combines trypsin-catalyzed (16)O/ (18)O labeling plus (16)O/ (18)O-methanol esterification for quantitation, a macro-immobilized metal-ion affinity chromatography trap for phosphopeptide enrichment, and LC-MS/MS analysis. LC separation and MS/MS are followed by neutral loss-dependent MS/MS/MS for phosphopeptide identification using a linear ion trap (LTQ)-FT mass spectrometer. A variety of phosphorylated proteins were identified and quantified including receptors, kinases, proteins associated with small GTPases, and cytoskeleton proteins. A number of hypothetical proteins were also identified as differentially expressed followed by LPA stimulation, and we have shown evidence of pseudopodia subcellular localization of one of these candidate proteins. These results demonstrate the efficiency of this quantitative phosphoproteomics approach and its application for rapid discovery of phosphorylation events associated with LPA gradient sensing and cell chemotaxis.     

Neutrophils augment recovery of porcine ischemia-injured ileal mucosa by an IL-1beta- and COX-2-dependent mechanism

Polymorphonuclear neutrophils (PMNs) play a critical role in intestinal mucosal injury and repair. To study effects of PMNs on acutely injured mucosa, we applied PMNs isolated from circulation or peritoneal fluid from animals with chemically induced peritonitis to ischemia-injured porcine ileal mucosa. In preliminary experiments, PMNs enhanced recovery of transepithelial electrical resistance (TER), and this action was inhibited by pretreatment with the nonselective cyclooxygenase (COX) inhibitor indomethacin. Because COX-2 is upregulated by inflammatory mediators such as IL-1beta, which is released by PMNs, we postulated that PMNs enhance recovery of ischemia-injured mucosa by a pathway involving IL-1beta and COX-2. Application of 5 x 10(6) PMNs to the serosal surface of ischemia-injured mucosa significantly enhanced recovery of TER (P < 0.05), an effect that was inhibited by the selective COX-2 inhibitor NS-398 (5 microM) and by an IL-1beta receptor antagonist (0.1 mg/ml). Addition of 10 ng/ml IL-1beta to the serosal surface of injured tissues caused a significant increase in TER (P < 0.05) that was inhibited by pretreatment with NS-398. Western blot analysis of mucosal homogenates revealed dramatic upregulation of COX-2 in response to IL-1beta or peritoneal PMNs, and the latter was inhibited by an IL-1beta receptor antagonist. Real-time PCR revealed that increased mRNA COX-2 expression preceded increased COX-2 protein expression in response to IL-1beta. We concluded that PMNs augment recovery of TER in ischemia-injured ileal mucosa via IL-1beta-dependent upregulation of COX-2.     

Production of nuclear transfer-derived swine that express the enhanced green fluorescent protein.

The ability to add or delete specific genes in swine will likely provide considerable benefits not just to agriculture but also to medicine, where pigs have potential as models for human disease and as organ donors. Here we have transferred nuclei from a genetically modified fibroblast cell line to porcine oocytes, matured in vitro under defined culture conditions, to create piglets expressing enhanced green fluorescent protein. The nuclear transfer-derived piglets were of normal size, although some mild symptoms of "large offspring syndrome" were evident. These experiments represent a next step towards creating swine with more useful genetic modifications.     

Trio Haploinsufficiency Causes Neurodevelopmental Disease-Associated Deficits

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