Cytoskeletal changes during neurogenesis in cultures of avian neural crest cells

Summary Neural crest cells are motile and mitotic, whereas their neuronal derivatives are terminally post-mitotic and consist of stationary cell body from which processes grow. The present study documents changes in the cytoskeleton that occur during neurogenesis in cultures of avian neural crest cells. The undifferentiated neural crest cells contain dense bundles of actin filaments throughout their cytoplasm, and a splayed array of microtubules attached to the centrosome. In newly differentiating neurons, the actin bundles are disrupted and most of the remaining actin filaments are reorganized into a cortical layer underlying the plasma membrane of the cell body and processes. Microtubules are more abundant in newly-differentiating neurons than in the undifferentiated cells, and individual microtubules can be seen dissociated from the centrosome. Neuron-specific -III tubulin appears in some crest cells prior to cessation of motility and cell division, and expression increases with total microtubule levels during neurogenesis. To investigate how these early cytoskeletal changes might contribute to alterations in morphology during neurogenesis, we have disrupted the cytoskeleton with pharmacologic agents. Microfilament disruption by cytochalasin immediately arrests the movement of neural crest cells and causes them to round-up, but does not significantly change the morphology of the immature neurons. Microtubule depolymerization by nocodazole slows the movement of undifferentiated cells and causes retraction of processes extended by the immature neurons. These results suggest that changes in the actin and microtubule arrays within neural crest cells govern distinct aspects of their morphogenesis into neurons.     

Highly diverse heparan sulfate analogue libraries: a novel resource for bioactivity screening of proteins

Introduction Fibroblast growth factors (FGFs) are a multipotent family of growth factors that are important for many biological processes, including development and wound healing. Normal, protease sensitive, prion protein (PrPC) can be converted to the protease resistant, infectious, form (PrPSc) believed to be associated with the pathogenesis of transmissible spongiform encephalopathies. FGFs signal through a family of tyrosine kinase receptors, the FGF receptors (FGFR) with the aid of heparan sulfate (HS), while the role of HS in the biology of PrPC is currently unknown, although depleting cells of HS can prevent production of PrPSc. HS, or its more highly sulfated relation heparin, can exert both positive and negative regulatory activities on a particular FGF‐FGFR combination. The nature of this regulation is determined by the structure of the HS that binds to the proteins. This structure is at least partially determined by the presence of particular sulfate groups along the sugar backbone. Identification of specific sulfate groups that can regulate the activity of proteins has been a long‐term goal in the field. Previously, heparins that had been completely lacking sulfates at specific positions were used to determine the binding and activity requirements for a particular protein. However, this may not necessarily allow for a full examination of the regulatory properties of HS. Here, we present a heparan sulfate analogue library produced by the partial, combinatorial desulfation of heparin. This library was the used to examine the structural properties of heparin required for FGF‐1 signalling through FGFR2c as well as the interactions of HS with PrPC. Materials and methods Porcine intestinal mucosal heparin was subjected to chemical desulfation and enzymatic cleavage. Polysaccharides and oligosaccharides derived from gel filtration chromatography and ion exchange chromatography were tested for their ability to activate FGF signalling through FGF receptors using a BaF3 assay system. Optical biosensors and cell assays were used to study the interaction of PrPC with chemically modified heparin. Results This library possessed vastly more heterogeneity than tissue heparan sulfates, allowing for more systematic screening to help identify those minimal structural features associated with activity. This library was used to examine the different structural features in heparin that support FGF‐1 signalling through FGFR2, showing that HS activity was not strictly dependant on size or charge. In addition, small, low‐sulfated heparins were found to interfere with the PrPC–heparin interaction. Discussion This supports the idea that overall structural features of the HS, rather than just the presence or absence of specific sulfate groups is important for the regulation of protein activity. Future efforts will be focused on further subfractionating the library and identifying specific structural features in HS that support FGF‐1 activity through FGFR2 and other FGFRs as well as the role of HS in the normal function of prion diseases, which may allow for the generation of novel, heparin‐based, therapeutics.     

Brief report: child-rearing practices of caregivers with and without a child with juvenile rheumatoid arthritis: perspectives of caregivers and professionals

OBJECTIVE: To evaluate predictions from professionals in pediatric rheumatology regarding the child-rearing practices of caregivers of children with juvenile rheumatoid arthritis (JRA) and healthy classmates. METHODS: Sixteen professionals identified items from the Child-Rearing Practices Report (CRPR) that were expected to differentiate between caregivers of children with JRA (64 mothers, 45 fathers) and caregivers of healthy classmates (64 mothers, 40 fathers). Families were interviewed, and physician ratings of disease severity were obtained. RESULTS: Experts predicted difficulties in protectiveness, discipline, and worry. Ratings from parents of children with JRA showed modest agreement with the professionals, surprising similarity to controls, and a limited association with disease factors. CONCLUSIONS: Contrary to expert opinion, JRA has only a modest influence on some child-rearing practices. Educating health care providers may minimize misperceptions about caring for children with JRA, and screening parents of children with more severe disease may assist in allocating education and services for families.     

A trade-off in replication in mosquito versus mammalian systems conferred by a point mutation in the NS4B protein of dengue virus type 4

An acceptable live-attenuated dengue virus vaccine candidate should have low potential for transmission by mosquitoes. We have identified and characterized a mutation in dengue virus type 4 (DEN4) that decreases the ability of the virus to infect mosquitoes. A panel of 1248 mutagenized virus clones generated previously by chemical mutagenesis was screened for decreased replication in mosquito C6/36 cells but efficient replication in simian Vero cells. One virus met these criteria and contained a single coding mutation: a C-to-U mutation at nucleotide 7129 resulting in a Pro-to-Leu change in amino acid 101 of the nonstructural 4B gene (NS4B P101L). This mutation results in decreased replication in C6/36 cells relative to wild-type DEN4, decreased infectivity for mosquitoes, enhanced replication in Vero and human HuH-7 cells, and enhanced replication in SCID mice implanted with HuH-7 cells (SCID-HuH-7 mice). A recombinant DEN4 virus (rDEN4) bearing this mutation exhibited the same set of phenotypes. Addition of the NS4B P101L mutation to rDEN4 bearing a 30 nucleotide deletion (Delta30) decreased the ability of the double-mutant virus to infect mosquitoes but increased its ability to replicate in SCID-HuH-7 mice. Although the NS4B P101L mutation decreases infectivity of DEN4 for mosquitoes, its ability to enhance replication in SCID-HuH-7 mice suggests that it might not be advantageous to include this specific mutation in an rDEN4 vaccine. The opposing effects of the NS4B P101L mutation in mosquito and vertebrate systems suggest that the NS4B protein is involved in maintaining the balance between efficient replication in the mosquito vector and the human host.     

Postmorten changes in the chemistry and histology of normal and edematous brains.

The brains of 18 patients were examined post mortem for histologic criteria of edema, and samples of white and gray matter were analyzed for water, sodium, and potassium content. In a parallel experimental study, brains of cats with unilateral freezing lesions and resulting cerebral edema were similarly examined immediately after death and up to 18 hours post mortem. In both types of material, in gray matter there was a relatively rapid (within less than 4 hours) increase in water and sodium content and fall in potassium content. In normal and edematous white matter, little change was observed post mortem. No correlation could be demonstrated in any of the material studied between water content and histologic grading for cerebral edema. It is concluded that determination of water content in the white matter postmortem could be a useful tool for the neuropathologist. Histologic assessment of cerebral edema is of little value.     

Distinct expression and localization of serine protease HtrA1 in human endometrium and first-trimester placenta.

Mammalian embryos cannot survive without the placenta. Development of the human placenta requires trophoblast proliferation, differentiation, and invasion as well as highly coordinated modulation of the maternal uterus. HtrA1 is a member of the recently identified mammalian HtrA (high temperature requirement factor A) serine protease family with a high level of expression in the placenta. In this study, we examined whether HtrA1 expression (mRNA and protein) is associated with placental development in the human. HtrA1 is up-regulated in both endometrial glands and decidual cells during endometrial preparation for embryo implantation and during first-trimester pregnancy at placentation. HtrA1 expression was also detected in certain trophoblast subtypes during early pregnancy. The villous syncytiotrophoblast and cytotrophoblast showed the strongest expression while the interstitial extravillous trophoblast showed the lowest or no expression of HtrA1. The distinct distribution of HtrA1 at the maternal-trophoblast interface suggests that HtrA1 may play a role in placental development.     

T cell-mediated immunity in the lung: a Cryptococcus neoformans pulmonary infection model using SCID and athymic nude mice.

T cells are important in systemic anticryptococcal defenses, but a role in controlling an initial pulmonary infection has not been demonstrated. A murine model with intratracheal inoculation was developed to study the acquisition and expression of pulmonary T cell-mediated immunity against Cryptococcus neoformans. Infections with four strains of C. neoformans (305, 68A, 613D, and 52D) in two strains of mice (BALB/c and C57BL/6) were examined. Unencapsulated strain 305 and slowly growing strain 68A were readily controlled apparently by nonimmune pulmonary defenses, and no extrapulmonary dissemination was detected. Strain 613D grew progressively in the lungs and disseminated to the brain and spleen. Strain 52D initially grew rapidly in the lungs and disseminated to the spleen, but a clearance mechanism developed in the lungs after day 7 postinfection and in the spleen after day 28. SCID and athymic nude mice were unable to clear a strain 52D pulmonary infection, and a lethal disseminated infection occurred. Pulmonary clearance could be adoptively transferred into SCID mice infected with strain 52D by use of immune T cells from the spleen and lungs and hilar lymph nodes of infected immunocompetent donors. Furthermore, pulmonary clearance was almost 100-fold better in SCID mice that received immune T cells from the lungs and hilar lymph nodes than in those that received immune T cells from the spleen, even though equivalent levels of delayed-type hypersensitivity were transferred by both cell populations. These adoptive transfer studies suggested that the lung and hilar lymph node T cells from immune animals either are enriched in such a way as to mediate protective immunity or home to the lungs better than do splenic T cells.     

Mitochondrial Ca(2+) buffering regulates synaptic transmission between retinal amacrine cells

The diverse functions of retinal amacrine cells are reliant on the physiological properties of their synapses. Here we examine the role of mitochondria as Ca(2+) buffering organelles in synaptic transmission between GABAergic amacrine cells. We used the protonophore p-trifluoromethoxy-phenylhydrazone (FCCP) to dissipate the membrane potential across the inner mitochondrial membrane that normally sustains the activity of the mitochondrial Ca(2+) uniporter. Measurements of cytosolic Ca(2+) levels reveal that prolonged depolarization-induced Ca(2+) elevations measured at the cell body are altered by inhibition of mitochondrial Ca(2+) uptake. Furthermore, an analysis of the ratio of Ca(2+) efflux on the plasma membrane Na-Ca exchanger to influx through Ca(2+) channels during voltage steps indicates that mitochondria can also buffer Ca(2+) loads induced by relatively brief stimuli. Importantly, we also demonstrate that mitochondrial Ca(2+) uptake operates at rest to help maintain low cytosolic Ca(2+) levels. This aspect of mitochondrial Ca(2+) buffering suggests that in amacrine cells, the normal function of Ca(2+)-dependent mechanisms would be contingent upon ongoing mitochondrial Ca(2+) uptake. To test the role of mitochondrial Ca(2+) buffering at amacrine cell synapses, we record from amacrine cells receiving GABAergic synaptic input. The Ca(2+) elevations produced by inhibition of mitochondrial Ca(2+) uptake are localized and sufficient in magnitude to stimulate exocytosis, indicating that mitochondria help to maintain low levels of exocytosis at rest. However, we found that inhibition of mitochondrial Ca(2+) uptake during evoked synaptic transmission results in a reduction in the charge transferred at the synapse. Recordings from isolated amacrine cells reveal that this is most likely due to the increase in the inactivation of presynaptic Ca(2+) channels observed in the absence of mitochondrial Ca(2+) buffering. These results demonstrate that mitochondrial Ca(2+) buffering plays a critical role in the function of amacrine cell synapses.     

Replication of a type I avian adenovirus (CELO) mutant in the chicken embryo

Summary Replication of a CELO large plaque (LP) mutant and that of its wild type small plaque (SP) parent was studied in the chorioallantoic membrane (CAM) and in the amnion of 11-day-old embryos. Although both strains produced essentially the same amount of virus in the tissue fluids, they differed in their rates of replication. Replication of the SP parent was maximal in the CAM 24 to 48 hours before that of the LP mutant. Whereas inclusions were observed in SP inoculated CAM 48 hours PI and were present during the course of study; LP inclusions were rare at 72 hours PI and thereafter; LP inclusions were seen at 72 hours PI. Fewer SP than LP particles were required to produce inclusions. No inclusions were seen in sections of the trachea and liver removed at 96 hours PI from embryos inoculated via the amniotic sac with LP and SP virus.Contribution 1466 of the Rhode Island Agriculture Experimental Station.