Arachidonate metabolites, platelet-activating factor, and the mobilization of protein kinase C in human polymorphonuclear neutrophils

In contrast to our previous report (Biochem. Biophys. Res. Comm. 134:587, 1986), we now find that protein kinase C (PKC) is mobilized in human polymorphonuclear neutrophils (PMN) stimulated with platelet-activating factor (PAF) or leukotriene (LT)B4. Thus nanomolar concentrations of each compound caused PMN to lose cytosolic, PKC-specific protein phosphorylating activity, as well as receptors for phorbol myristate acetate (PMA). Smaller gains in membrane-associated PMA receptors accompanied these changes. Diacylglycerol and PMA had very similar effects on PKC. However, unlike these direct PKC activators, PAF and LTB4 induced only moderate decreases in cytosolic PKC; acted only on PMN pretreated with cytochalasin B; did not mobilize PKC in disrupted PMN or activate PKC in a cell-free system; and with respect to PAF, induced responses that partially reversed within 30 min. Furthermore, PAF, LTB4, and several of their structural analogues mobilized PKC at concentrations correlating closely with their respective affinities for cellular LTB4 or PAF receptors. Thus PAF and LTB4 acted by indirect and apparently receptor-mediated mechanisms. Four observations indicated that the cytochalasin B-dependent degranulating actions of PAF and LTB4 involved PKC. First, PKC mobilization and degranulation occurred at the same stimulus concentrations. Second, 5-hydroxyicosatetraenoate dramatically enhanced both PKC mobilization and degranulation when elicited by PAF; it had relatively little influence on LTB4-induced responses. Third, PAF-induced mobilization (t1/2 less than 7 sec) preceded degranulation (t1/2 approximately 20 sec). Finally, a PKC blocker, polymyxin B, was similarly effective in inhibiting degranulation responses to PAF, LTB4, and PMA. Because stimulated PMN may produce and use PAF, LTB4, and 5-hydroxyicosatetraenoate as secondary intracellular mediators, our results implicate PKC as a central and potentially critical regulator of function.     

Levels of protein kinase C activity in human gastrointestinal cancers

The protein kinase C (PKC) activities of tumor tissue and adjacent normal mucosa of human cancers of the esophagus (8 cases), stomach (1 case) and colon (3 cases) were measured. Considerable variations were found in the activity of PKC and in its subcellular distribution in these cancers. The PKC activities of the membrane and cytosolic fractions of the eight esophageal cancers were, however, similar to those of the adjacent normal mucosa: the average PKC activities of the tumor tissues and normal mucosa were 7.5 and 8.3 pmol/min/mg protein, respectively, in their membrane fractions and 7.9 and 7.8 pmol/min/mg protein, respectively, in their cytosolic fractions.     

Circular dichroic evidence for regulation of enzymatic activity by nonsubstrate hydrophobic ligand on glutathione S-transferase P.

1-Anilinonaphthalene-8-sulfonic acid (ANS) noncompetitively inhibited enzyme activity of glutathione S-transferase P for both glutathione and 1-chloro-2,4-dinitrobenzene (Ki = 30 microM). Dissociation constant for ANS.GST-P complex calculated from the binding study was 15 microM. From the similar values of the inhibition constant and the dissociation constant, it was concluded that specific ANS binding caused the loss of enzyme activity. In the protein structural analysis by circular dichroism, the secondary structures remarkably changed by ANS binding in accordance with the decrease of enzymatic activities. The conformational change of the protein and the decrease in enzymatic activity were reversed by dissociation of ANS. This fact strongly suggested that the enzymatic activity was regulated by a nonsubstrate hydrophobic ligand.     

Threshold of the soleus muscle H-reflex is less sensitive to the change in excitability of the motoneuron pool during plantarflexion or dorsiflexion in humans

In the present study, we investigated whether weak (10% of maximal voluntary contraction) tonic dorsiflexion (DF) and plantarflexion (PF) affects the two conventional parameters used for evaluating the excitability of the soleus motoneuron (MN) pool, i.e. the ratio of the threshold of H-reflex to that of M-response (H_th:M_th) and the ratio of the maximal amplitude of H-reflex to that of M-response (H_max:M_max) in human subjects. The results showed that the H_max:M_max decreased during DF and increased during PF compared with that during rest, whereas no clear alteration was observed in H_th:M_th. These results are consistent with the scheme proposed by earlier workers, who have argued that neither inhibitory nor facilitatory effects of the conditioning stimulus apply to specific spinal reflex circuits occurring around the threshold of the test H-reflex. It is suggested, therefore, that the conventional use of the H_th:M_th ratio as a parameter reflecting the excitability of the MN pool should be reconsidered.     

Histochemical Demonstration of Enzyme Activities in Plastic and Paraffin Embedded Tissue Sections

Histochemical staining for enzymes is usually performed on frozen sections. This report lists the longer incubation times required to demonstrate esterase, acid phosphatase, β-galactosidase, and cytochrome oxidase in plastic embedded and routine paraffin embedded tissues. The sections embedded in plastic, i.e. water soluble methacrylate (Polyscience's JB-4) and cut at 2 μm, were far superior to frozen Sections and paraffin embedded sections both in tissue detail and in the localization of the histochemical reaction product.     

Theoretical evaluation of a model of the catalytic triads of serine and cysteine proteases by ab initio molecular orbital calculation

Serine and cysteine proteases are structurally distinct families of proteolytic enzymes that exert analogous catalytic reactions. It is known that serine, histidine, and aspartate residues are involved in a putative catalytic triad of serine proteases whereas cysteine, histidine and aspartate (or asparagine) are involved in that of cysteine proteases. Although the overall three-dimensional structures of the two classes of proteases are quite different, a similar feature of the catalytic mechanism can be observed. We carried out a theoretical study in the gas phase on their two catalytic steps using ab initio molecular orbital calculation to evaluate the efficiency of each proteolytic activity. To examine the key aspects of the catalytic processes in their active sites, we employed simple molecular models, in which serine and histidine were substituted for by CH3OH and CH3SH, respectively, and aspartate by CH3CO2-. Molecular geometries of the two models were fully optimized by an energy-gradient method. The activation energies for proton transfer in the proteolytic steps and the total energies of each molecule in the intermediate states were calculated. These results revealed that the activation energy for proton transfer in the CH3SH-His-CH3CO2-system was smaller by 11 kcal mol-1than that of the CH3OH-His-CH3CO2-system. The calculated potential energy surface and the relative magnitudes of all steps in these reaction paths with regard to double proton transfer supported the above results. Taken together, these results indicated that the Cys-His-Asp proteolytic system might work more efficiently than the Ser-His Asp system.     

Histochemical analysis of macrophage migration inhibitory factor in psoriasis vulgaris

Psoriasis is a persistent cutaneous disease characterized by skin inflammation and infiltration of immunocytes such as lymphocytes and monocytes/macrophages, concomitant with abnormal epidermal hyperproliferation. We previously showed that the serum level of macrophage migration inhibitory factor (MIF) and its production by peripheral blood mononuclear cells of patients with psoriasis were closely correlated with the severity of clinical symptoms; however, the precise role of MIF in psoriatic epidermis remains to be clarified. The current study was carried out to elucidate the possible involvement of MIF in psoriasis, using immunohistochemistry and in situ hybridization. In contrast to elevated serum MIF in psoriasis, MIF-positive staining in the lesional psoriatic epidermis was significantly decreased, as demonstrated by immunohistochemical analysis using an anti-MIF antibody. Consistent with this finding, we found, by in situ hybridization, that MIF mRNA concomitantly decreased in the psoriatic lesions. Although the reason for the different MIF levels in the psoriatic epidermis and in the circulation remains unknown, it is hypothesized that MIF, a potential growth factor, might be decreased in psoriatic lesions to counterregulate the abnormal epidermal proliferation caused by dysregulation of cytokines and growth factors.     

Role of macrophage migration inhibitory factor (MIF) in peripheral nerve regeneration: anti-MIF antibody induces delay of nerve regeneration and the apoptosis of Schwann cells

BACKGROUND: Macrophage migration inhibitory factor (MIF) is a pluripotent cytokine involved in inflammation and immune responses as well as in cell growth. Although we previously demonstrated the presence of MIF in peripheral nerves, and MIF mRNA expression was up-regulated after axotomy, the role of MIF in nerve injury and regeneration has not been evaluated. MATERIALS AND METHODS: To examine the potential role of MIF in nerve regeneration, we locally administered an anti-MIF polyclonal antibody into regenerating rat sciatic nerves using the silicone chamber model. The effect of the anti-MIF antibody on nerve regeneration was evaluated using an axonal reflex test. In addition, we carried out a terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) assay and immunohistochemical analysis of the damaged nerve segments with regard to apoptosis-related proteins such as p53 to evaluate the effects of anti- MIF antibodies on apoptosis during the regeneration process. RESULTS: The regeneration length of the nerve in the anti-MIF antibody-treated group was significantly shorter than that in the non-immune rabbit IgG-treated group at weeks 2, 4 and 6 after surgery. TUNEL assay showed that a large number of apoptotic cells, mostly Schwann cells, were observed in the intratubal and distal nerve segments at weeks 4 and 6 after surgery by the anti-MIF antibody treatment. Consistent with these results, Ki-67-positive cells were significantly decreased by the anti-MIF antibody treatment. Immunohistochemical analyses revealed that p53 and, to a lesser extent, Fas were more up-regulated in the anti-MIF antibody-treated nerves than in the controls. CONCLUSION: Taken together, these results suggest that MIF plays an important role in acceleration of peripheral nerve regeneration and in prevention of Schwann cell apoptosis, mainly through overcoming the apoptotic effect of p53.