Contribution of Corneal Neovascularization to Dendritic Cell Migration into the Central Area during Human Corneal Infection

Compared with the peripheral corneal limbus, the human central cornea lacks blood vessels, which is responsible for its immunologically privileged status and high transparency. Dendritic cells (DCs) are present in the central avascular area of inflamed corneas, but the mechanisms of their migration to this location are poorly understood. Here, we investigated the contribution of vessel formation to DC migration into the central cornea, and analyzed the DC chemotactic factors produced by human corneal epithelial (HCE) cells. Using human eyes obtained from surgical procedures, we then assessed vessel formation, DC distribution, and activin A expression immunohistochemically. The results demonstrated increased numbers of vessels and DCs in the central area of inflamed corneas, and a positive correlation between the number of vessels and DCs. Activin A was expressed in the subepithelial space and the endothelium of newly formed blood vessels in the inflamed cornea. In infected corneas, DCs were present in the central area but no vascularization was observed, suggesting the presence of chemotactic factors that induced DC migration from the limbal vessels. To test this hypothesis, we assessed the migration of monocyte-derived DCs toward HCE cell supernatants with or without lipopolysaccharide (LPS) stimulation of HCE cells and inflammatory cytokines (released by HCE cells). DCs migrated toward tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, and activin A, as well as LPS-stimulated HCE cell supernatants. The supernatant contained elevated TNF-α, IL-6, and activin A levels, suggesting that they were produced by HCE cells after LPS stimulation. Therefore, vessels in the central cornea might constitute a DC migration route, and activin A expressed in the endothelium of newly formed vessels might contribute to corneal vascularization. Activin A also functions as a chemotactic factor, similar to HCE-produced TNF-α and IL-6. These findings enhance our understanding of the pathophysiology of corneal inflammation during infection.     

'Protected DNA Probes' capable of strong hybridization without removal of base protecting groups

We propose a new strategy called the ‘Protected DNA Probes (PDP) method’ in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac⁴C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac⁶az⁸c⁷A). It was found that PDP containing ac⁴C and ac⁶az⁸c⁷A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness.     

Ingested beta-carotene enhances glutathione level and up-regulates the activity of cysteine cathepsin in murine splenocytes

To elucidate health benefits of beta-carotene, especially on immunity, we measured redox-related indices in spleen cells from BALB/c mice supplemented with various amounts of beta-carotene. In mice supplemented with beta-carotene in their diet, glutathione, an intracellular anti-oxidation agent, increased in their splenocytes. This change was highly correlated with the accumulation of beta-carotene, but not with that of retinol. The increase in glutathione was accompanied by an increase in mRNA for gamma-glutamylcysteine synthetase, a rate-limiting enzyme for glutathione synthesis. The higher the glutathione content was in the spleen cells, the higher the activity of cysteine cathepsin became in crude antigen-presenting cells contained in the spleen. These data suggest that accumulated beta-carotene in splenocytes, without being metabolized, caused an increase in the intracellular glutathione level, thereby anti-oxidatively supporting the activity of redox-sensitive lysosomal protease, which is involved in antigen-presentation.     

Characterization of conformational deformation-coupled interaction between immunoglobulin G1 Fc glycoprotein and a low-affinity Fcγ receptor by deuteration-assisted small-angle neutron scattering

A recently developed integrative approach combining varied types of experimental data has been successfully applied to three-dimensional modelling of larger biomacromolecular complexes. Deuteration-assisted small-angle neutron scattering (SANS) plays a unique role in this approach by making it possible to observe selected components in the complex. It enables integrative modelling of biomolecular complexes based on building-block structures typically provided by X-ray crystallography. In this integrative approach, it is important to be aware of the flexible properties of the individual building blocks. Here we examine the ability of SANS to detect a subtle conformational change of a multidomain protein using the Fc portion of human immunoglobulin G (IgG) interacting with a soluble form of the low-affinity Fcγ receptor IIIb (sFcγRIIIb) as a model system. The IgG-Fc glycoprotein was subjected to SANS in the absence and presence of 75%-deuterated sFcγRIIIb, which was matched out in D₂O solution. This inverse contrast-matching technique enabled selective observation of SANS from IgG-Fc, thereby detecting its subtle structural deformation induced by the receptor binding. The SANS data were successfully interpreted by considering previously reported crystallographic data and an equilibrium between free and sFcγRIIIb-bound forms. Our SANS data thus demonstrate the applicability of SANS in the integrative approach dealing with biomacromolecular complexes composed of weakly associated building blocks with conformational plasticity.     

Transduction of R plasmids by bacteriophages P1 and P22

Summary We demonstrated that bacteriophages P1 and P22 were able to form various types of hybrids with six out of seven different R plasmids tested. When the same R plasmid was used for isolation, P1-R hybrids usually carried more resistance markers than P22-R. Several genetical observations suggest that the hybrid prophages carried the resistance markers transposed to the phage genomes without loss of essential phage genes. Upon UV-irradiation the prophages produced phage lysates that transduced the relevant resistance markers at high frequencies by lysogenic conversion. The insertion of the resistance markers was even acquired by the P1 or P22 genomes during one-step growth in R⁺ cells. Some lytically prepared lysates grown on R⁺ cells contained the hybrids at a frequency of 10^-7 to 10^-6/plaqueforming unit. Analysis of P1-transductants for resistance markers of the R plasmids revealed in some cases more specialized transductants than generalized transductants. These results strongly indicate that a precise genetic map of an R plasmid can not be established only on the basis of co-transduction frequencies of the resistance markers of the R plasmid.     

Myocardial Dysfunction Induced by Food Restriction is Related to Morphological Damage in Normotensive Middle-Aged Rats

Summary Previous works from our laboratory have revealed that food restriction (FR) promotes discrete myocardial dysfunction in young rats. We examined the effects of FR on cardiac function, in vivo and in vitro, and ultrastructural changes in the heart of middle-aged rats. Twelve-month-old Wistar-Kyoto rats were fed a control (C) or restricted diet (daily intake reduced to 50% of the control group) for 90 days. Cardiac performance was studied by echocardiogram and in isolated left ventricular (LV) papillary muscle by isometric contraction in basal condition, after calcium chloride (5.2 mM) and beta-adrenergic stimulation with isoproterenol (10⁻⁶ M). FR did not change left ventricular function, but increased time to peak tension, and decreased maximum rate of papillary muscle tension development. Inotropic maneuvers promoted similar effects in both groups. Ultrastructural alterations were seen in most FR rat muscle fibers and included, absence and/or disorganization of myofilaments and Z line, hyper-contracted myofibrils, polymorphic and swollen mitochondria with disorganized cristae, and a great quantity of collagen fibrils. In conclusion, cardiac muscle sensitivity to isoproterenol and elevation of extracellular calcium concentration is preserved in middle-aged FR rats. The intrinsic muscle performance depression might be related to morphological damage.     

Heterozygous knockout of neuregulin-1 gene in mice exacerbates doxorubicin-induced heart failure

Neuregulins and their erbB receptors are essential for cardiac development and postulated to be cardioprotective in the presence of injury in the postnatal heart. We tested the hypothesis that the development of doxorubicin-induced cardiotoxicity in vivo is more severe in mice with heterozygous knockout of the neuregulin-1 gene (NRG-1(+/-)) compared with wild-type mice (WT). Three-month old NRG-1(+/-) and WT mice were injected with a single dose of doxorubicin (20 mg/kg ip). Survival was analyzed by the Kaplan-Meier approach. Left ventricular (LV) function and signaling pathways were analyzed 4 days after treatment. Fifteen days after treatment, survival was significantly lower in doxorubicin-treated NRG-1(+/-) mice (NRG-1(+/-)-Dox) compared with doxorubicin-treated WT mice (WT-Dox) (15% vs. 33%, P < 0.01). LV mass was significantly lower in NRG-1(+/-)-Dox but not in WT-Dox compared with nontreated animals. LV systolic pressure and LV midwall fractional shortening were significantly lower in NRG-1(+/-)-Dox compared with WT-Dox mice. LV protein levels of NRG-1, erbB2, and erbB4 receptors were similar in WT-Dox and NRG-1(+/-)-Dox mice. However, levels of phosphorylated erbB2, Akt, and ERK-1/2 were significantly decreased in NRG-1(+/-)-Dox compared with WT-Dox mice. A significant decrease in phosphorylated P70S6K levels was also observed in NRG-1(+/-)-Dox compared with nontreated NRG-1(+/-) mice. These results demonstrate that heterozygous knockout of the neuregulin-1 gene worsens survival and LV function in the presence of doxorubicin-induced cardiac injury in vivo. This is associated with the depression of activation of the erbB2 receptor as well as Akt, p70S6K, and ERK-1/2 pathways.     

Occurrence of Chloramphenicol-acetylating Enzymes in Various Gram-negative Bacilli

The occurrence of a chloramphenicol-acetylating enzyme, similar to that found in Escherichia coli, carrying an R factor was investigated in various gram-negative bacilli. The acetylated products of chloramphenicol were identified by chromatography and quantitatively assayed after benzene extraction. The investigated strains were of the Salmonella-Arizona group, the Klebsiella-Aerobacter group, Serratia marcescens, the Proteus group, and Pseudomonas aeruginosa, most of which were isolated from 1947 to 1957. Both chloramphenicol-sensitive and -resistant strains were included, but none of them was able to transfer chloramphenicol resistance by conjugation. In the Proteus group, a significant level of a chloramphenicol-acetylating enzyme was found in most strains, whether they were sensitive or resistant to chloramphenicol; the resistant strains showed higher levels of the enzyme. Some chloramphenicol-sensitive strains lacked this enzyme. Only the sensitive strains containing the enzyme could easily produce chloramphenicol-resistant mutants with higher enzyme activity. Thus, the chloramphenicol resistance of this group can be reasonably explained on the basis of the chloramphenicol-acetylating enzyme. All of the Pseudomonas aeruginosa strains were resistant to chloramphenicol, and most strains showed low levels of the enzyme (which, however, did not appear sufficient to explain their resistance). All of the strains of the other groups (except one strain of Enterobacter cloacae) lacked the enzyme, although most strains of the Klebsiella-Aerobacter group and of S. marcescens were resistant to chloramphenicol. With respect to the origin of the resistance gene of the R factor, it is noteworthy that the strains of Proteus mirabilis isolated in 1947 possessed this enzyme before the discovery of chloramphenicol.     

Effects of reserpine on dopamine metabolite in the nucleus accumbens and locomotor activity in freely moving rats

The effect of reserpine (2 mg/kg i.p.) on both locomotor activity and the turnover of dopamine metabolite in the rat nucleus accumbens was estimated by using an activity monitor (Animex) and by in vivo brain microdialysis. Three to five hours after reserpine administration locomotor activity was reduced and there was a concomitant increase in the level of the dopamine metabolite, homovamillic acid. These findings suggest that depletion of dopamine from the nucleus accumbens may result in decreased locomotor activity. The data support the notion that dopamine in this tissue contributes to the control of locomotion.