No abstract

Since NO production by NOS-2 made by astrocytes activated by proinflammatory cytokines contributes to the killing of neurons in variously damaged human brains, knowing the mechanisms responsible for NOS-2 expression should contribute to developing effective therapeutics. The expression and activation of NOS-2 in normal adult human cerebral cortical astrocytes treated with three proinflammatory cytokines, IL-1beta, TNF-alpha, and IFN-gamma, are driven by two separable mechanisms. NOS-2 expression requires a burst of p38 MAPK activity, while the activation of the resulting enzyme protein requires MEK/ERK-dependent BH4 (tetrahydrobiopterin) synthesis between 24 and 24.5 h after adding the cytokines to the culture medium. Here we show that NOS-2 expression in the activated astrocytes requires that the culture medium contain 1.8 mM Ca2+, but it is unaffected by inhibiting calcium-sensing receptors (CASRs) with NPS 89636. However, NOS-2 activation is inhibited by NPS 89626 during the MEK/ERK-dependent stage between 24 and 24.5 h after adding the cytokines, and this inhibition can be overridden by exogenous BH4. Therefore, NOS-2 expression and the subsequent BH4-dependent NOS-2-activation in human astrocytes need 1.8 mM Ca2+ to be in the culture medium, while NOS-2 activation also needs functional CASRs between 24 and 24.5 h after cytokine addition. These findings raise the possibility that calcilytic drugs prevent NO-induced damage and death of human neurons.

No abstract

The role of protein kinase C-␤ II (PKC-␤ II ) in etoposide (VP-16)-induced apoptosis was studied using polyomavirus-transformed pyF111 rat fibroblasts in which PKC-␤ II specific activity in the nuclear membrane (NM) doubled and the enzyme was cleaved into catalytic fragments. No PKC-␤ II complexes with lamin B1 and/or active caspases were immunoprecipitable from the NM of proliferating untreated cells, but large complexes of PKC-␤ II holoprotein and its catalytic fragments with lamin B1, active caspase-3 and -6, and inactive phospho-CDK-1, but not PKC-␤ I or PKC-␦, could be immunoprecipitated from the NM of VP-16-treated cells, suggesting that PKC-␤ II is an apoptotic lamin kinase. By 30 min after normal nuclei were mixed with cytoplasms from VP-16-treated, but not untreated, cells, PKC-␤ II holoprotein had moved from the apoptotic cytoplasm to the normal NM, and lamin B1 was phosphorylated before cleavage by caspase-6. Lamin B1 phosphorylation was partly reduced, but its cleavage was completely prevented, despite the presence of active caspase-6, by adding a selective PKC-␤s inhibitor, hispidin, to the apoptotic cytoplasms. Thus, a PKC-␤ II response to VP-16 seems necessary for lamin B1 cleavage by caspase-6 and nuclear lamina dissolution in apoptosing pyF111 fibroblasts. The possibility of PKC-␤ II being an apoptotic lamin kinase in these cells was further suggested by lamin B1-bound PKC-␦ being inactive or only slightly active and by PKC-␣ not combining with the lamin.A growing body of evidence suggests that the commitment to and execution of apoptosis are mediated through the phosphorylation of specific proteins by several protein kinases (1). A number of PKC 1 isoforms appear to be among these protein kinases (2, 3). Apoptogens may activate or inactivate and cause the translocation of various PKC isoforms from the cytosol onto cytoskeletal components, cytoplasmic membranes, mitochondria, and/or the nuclear envelope (4 -11); induce their migration from such subcellular structures to nucleoplasmic and/or cytosolic fractions (8, 12); or cause the PKC holoenzymes to be cleaved into N-terminal regulatory and C-terminal catalytic fragments (CFs) by several proteases, including caspases (11,(13)(14)(15). Despite suggestions that a particular PKC isoform (e.g. the novel PKC-␦ and/or its CFs) might play a pivotal role in apoptosis (13,14), the available evidence indicates the involvement and cleavage of novel PKCs (e.g. PKC-⑀ and PKC-), the classical Ca 2ϩ -stimulable PKCs (e.g. PKC-␤), atypical PKCs (e.g. PKC-), and PKC-related (e.g. PRK) kinases in apoptosis (2,3,15). This is the third report from a continuing study of the roles of protein kinase C isozymes in drug-induced apoptosis using a polyomavirus-transformed embryo rat fibroblast, the pyF111 cell, as a model (10, 11). We chose this fibroblast, since it is prone to apoptosis because it cannot make the antiapoptotic Bcl-2 and Bcl-X L proteins but can make the proapoptotic Bax protein (10, 11). We have shown that whereas a surge of the activity of PKC-␦ holopr...

Biologically well-tolerated materials are at the core of intense research. In this study, we report both the intrinsic features of formic acid (FA)-crosslinked threedimensional (3D) nonwoven scaffolds consisting of sericindeprived, Bombyx mori silk fibroin (SF) in ß-sheet form and the long-term co-culture of adult human epidermal keratinocytes (HEKs) and dermal fibroblasts (HDFs) on them. The results of scanning electron microscopy, differential scanning calorimetry, and thermogravimetric and tensile strength studies showed that such scaffolds are made by a composite material, in which anisotropic SF fibres are enclosed within an isotropic matrix of SF in film form. Both fibres and films are firmly crosslinked by FA treatment and water-insoluble owing to their ß-sheet crystalline structure. Moreover, while a certain strength of the material in the dry state may favour its manufacturing, handling, and shaping, the dramatic loss of hardiness under wet conditions renders the scaffolds softer, mechanically more compliant and, hence, more apt for implantation. Normal HEKs and HDFs could be successfully co-cultured on such nonwovens for up to 75-95 days in vitro, thus forming a novel kind of dermo-epidermal equivalent, in which the cells were metabolically active and performed specific functions (e.g. the de novo production and assembly of collagen fibres), but never released urea nitrogen (an index of protein catabolism) or proinflammatory interleukin-1ß (IL-1ß). In keeping with the results of previous studies in animals (Dal Pra I, et al, Biomaterials 26: 1987-1989, 2005), these findings support the view that 3D SF-based nonwovens may be excellent candidates for beneficial applications in the field of human tissue engineering/regeneration/repair.