Immunohistochemical study on Fas and Fas ligand in skin wound healing

An immunohistochemical study on the expression of Fas and Fas ligand (Fas L) was performed in order to examine the role of apoptosis through Fas–Fas L in mouse skin wound healing. After a 1-cm-long incision in the central dorsum skin, mice were sacrificed at intervals ranging from 0.5 to 240h, followed by the sampling of wound margin. The expression of Fas and Fas L in the wound margins and in uninjured skin controls was studied using frozen sections. In uninjured skin controls, a very weak expression of Fas and Fas L was detected immunohistochemically in hair follicles, sebaceous glands and epidermal cells. In wounded specimens, polymorphonuclear cells and inflammatory mononuclear ones (round-shaped and spindle-shaped types) were evident. A single immunostaining showed that Fas or Fas L was detectable in inflammatory mononuclear cells involved in the skin wound healing process. Double immunostaining for Fas and Fas L revealed that inflammatory mononuclear cells co-expressed both antigens. In situ TUNEL combined with immunostaining showed that the inflammatory mononuclear cells expressing Fas or Fas L and the polymorphonuclear cells were TUNEL-stained, although neither Fas nor Fas L was detected in the polymorphonuclear cells. The number of TUNEL-positive, inflammatory mononuclear cells expressing Fas or Fas L per 0.01×0.01cm² was counted. The average number of 10 randomly selected microscope fields reached a peak at the fibro-proliferative phase of wound healing. These results indicate that apoptosis through Fas and Fas L may play an important role for reducing the cellularity during skin wound healing in mice.     

Central projections of the wing afferents in the hawkmoth, Agrius convolvuli

Flight behaviors in various insect species are closely correlated with their mechanical and neuronal properties. Compared to locusts and flies which have been intensively studied, moths have “intermediate” properties in terms of the neurogenic muscle activations, power generation by indirect muscles, and two-winged-insect-like flapping behavior. Despite these unique characteristics, little is known about the neuronal mechanisms of flight control in moths. We investigated projections of the wing mechanosensory afferents in the central nervous system (CNS) of the hawkmoth, Agrius convolvuli, because the mechanosensory proprioceptive feedback has an essential role for flight control and would be presumably optimized for insect species. We conducted anterograde staining of nine afferent nerves from the fore- and hindwings. All of these afferents projected into the prothoracic, mesothoracic and metathoracic ganglia (TG1, 2 and 3) and had ascending fibers to the head ganglia. Prominent projection areas in the TG1–3 and suboesophageal ganglion (SOG) were common between the forewing, hindwing and contralateral forewing afferents, suggesting that information from different wings are converged at multiple levels presumably for coordinating wing flapping. On the other hand, differences of projections between the fore- and hindwing afferents were observed especially in projection areas of the tegulae in the TG1 and contralateral projections of the anterior forewing nerve in the TGs and SOG, which would reflect functional differences between corresponding mechanoreceptors on each wing. Afferents comprising groups of the campaniform sensilla at the wing bases had prominent ascending pathways to the SOG, resembling the head–neck motor system for gaze control in flies. Double staining of the wing afferents and flight or neck motoneurons also indicated potential connectivity between them. Our results suggest multiple roles of the wing proprioceptive feedback for flight and provide the anatomical basis for further understanding of neuronal mechanisms of the flight system in moths.     

Cessation of Fgf10 signaling, resulting in a defective dental epithelial stem cell compartment, leads to the transition from crown to root formation

Mouse, rat and human molars begin to form root after the completion of crown formation. In these teeth, fibroblast growth factor (Fgf) 10 disappears in the transitional stage from crown formation to root. By contrast, rodent incisors and vole molars demonstrate continuous growth, owing to the formation and maintenance of a stem cell compartment by the constant expression of Fgf10. To clarify the relationship between root formation and disappearance of Fgf10, we carried out two experiments for the loss and gain of Fgf10 function. First, we examined postnatal growth in the incisors of Fgf10-deficient mice, which have the defect of a dental epithelial stem cell compartment referred to as ;apical bud', after implantation under the kidney capsule. The growth at the labial side in the mutant mice mimics the development of limited-growth teeth. 5'-Bromo-2'-deoxyuridine (BrdU) labeling and cytokeratin (CK) 14 and Notch2 immunostaining suggested that the inhibition of inner enamel epithelium growth and the more-active proliferation of the outer enamel epithelium and/or stellate reticulum result in Hertwig's epithelial root sheath formation. Second, we examined the effects of Fgf10 overexpression in the transitional stage of molar germs, which led to the formation of apical bud involving in the inhibition of HERS formation. Taken together, these results suggest that the disappearance of Fgf10 signaling leads to the transition from crown to root formation, owing to the loss of a dental epithelial stem cell compartment.     

Novel products generated from 2'-deoxyguanosine by hypochlorous acid or a myeloperoxidase-H2O2-Cl- system: identification of diimino-imidazole and amino-imidazolone nucleosides

Hypochlorous acid (HOCl), generated by myeloperoxidase from H2O2 and Cl-, plays an important role in host defense and inflammatory tissue injury. We report here the identification of products generated from 2'-deoxyguanosine (dGuo) with HOCl. When 1 mM dGuo and 1 mM HOCl were reacted at pH 7.4 and 37 degrees C for 15 min and the reaction was terminated with N-acetylcysteine (N-AcCys), two products were generated in addition to 8-chloro-2'-deoxyguanosine (8-Cl-dGuo). One was identified as an amino-imidazolone nucleoside (dIz), a previously reported product of dGuo with other oxidation systems. The other was identified as a novel diimino-imidazole nucleoside, 2,5-diimino-4-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-2H,5H-imidazole (dDiz) by spectrometric measurements. The yields were 1.4% dDiz, 0.6% dIz and 2.4% 8-Cl-dGuo, with 61.5% unreacted dGuo. Precursors of dDiz and dIz containing a chlorine atom were found in the reaction solution in the absence of termination by N-AcCys. dDiz, dIz and 8-Cl-dGuo were also formed from the reaction of dGuo with myeloperoxidase in the presence of H2O2 and Cl- under mildly acidic conditions. These results imply that dDiz and dIz are generated from dGuo via chlorination by electrophilic attack of HOCl and subsequent dechlorination by N-AcCys. These products may play a role in cytotoxic and/or genotoxic effects of HOCl.     

Identification of a plausible biosynthetic enzyme for the IM-2-type autoregulator in Streptomyces antibioticus

Virginiae butanolides (VBs) and IM-2 are members of Streptomyces hormones called ‘butyrolactone autoregulators’ which regulate the antibiotic production in Streptomyces species at nanomolar concentrations. Cell-free extract of a VB-A overproducer, Streptomyces antibioticus NF-18, is capable of catalyzing the final step of the autoregulator biosynthesis, namely, the NADPH-dependent reduction of 6-dehydroVB-A. However, physico-chemical analyses of the purified enzymatic products revealed that, in addition to the VB-type isomer [(2R,3R,6S)-enantiomer], IM-2-type isomers [(2R,3R,6R)- and (2S,3S,6S)-enantiomers] were also produced from (±)-6-dehydroVB-A, suggesting the existence of several 6-dehydroVB-A reductases with respective stereoselectivities. The reductase activity of the crude extracts was separated into two activity peaks, peak I (major) and peak II (minor), by DEAE-5PW HPLC. Chiral HPLC analyses demonstrated that peak I enzyme and peak II enzyme catalyzed the production of (2R,3R,6S), (2R,3R,6R) and (2S,3S,6S) isomers at ratios of 46:1:3.2 and 4.9:1:1.5, respectively, indicating clearly that S. antibioticus NF-18 possesses at least two 6-dehydroVB-A reductases: one much favored toward VB-A biosynthesis, the other with relaxed stereoselectivity capable of synthesizing both VB-type and IM-2-type autoregulators.     

Alpha-interferon in Kikuchi's disease.

In Japan, histiocytic necrotizing lymphadenitis (Kikuchi's disease) is a relatively common reactive lesion affecting lymph nodes, but the histogenesis and pathogenesis of the disease have not been clarified. Alpha-interferon has a role in the body's defense against viral infections. Using a polyclonal antibody to human alpha-interferon, we found numerous cells, mainly histiocytes, containing alpha-interferon in affected foci in the lymph nodes from 24 patients with Kikuchi's disease. Tubuloreticular structures, thought by some authors to be associated with the production of interferon, were detected by electron microscopy in histiocytes, activated lymphocytes and vascular endothelial cells in the affected foci. These results suggested that the formation of tubuloreticular structures is a secondary phenomenon following stimulation by alpha-interferon. Further, the activity of 2'-5' oligoadenylate synthetase, which is induced by alpha-interferon and enhanced during the early or active stage of viral infection, showed increased levels of activity in the active stage of Kikuchi's disease and decreased to normal levels in the convalescent stage 2 weeks later. These results suggested the possibility of a viral etiology for Kikuchi's disease.     

Isoelectric point of an ion-penetrable membrane.

The surface potential of an ion-penetrable planar membrane is calculated for the case in which acidic and basic groups are present in the membrane. It is found that when both acidic and basic groups are not uniformly distributed in the direction normal to the membrane, the isoelectric point (the pH value at which the surface potential becomes zero) of the membrane varies with the electrolyte concentration, whereas if both groups are uniformly distributed, the isoelectric point is independent of the electrolyte concentration. As a simple example, we treat a membrane consisting of two layers, in which acidic groups are distributed in the outer layer and basic groups are in the inner layer. Simple equations determining the membrane surface potential as a function of pH and electrolyte concentration and the dependence of the isoelectric point on the electrolyte concentration are presented.