Angiogenesis in the mouse retina: a model system for experimental manipulation

The vascular system of the mouse retina provides a useful model for analyzing the molecular and cellular mechanisms regulating angiogenesis because (1) hierarchical vascular networks are newly formed only after birth, (2) the cellular components involved in angiogenesis are well characterized, and (3) all the processes are accessible for monitoring and manipulation. In this article, we present an overview of our current understanding of the process of retinal angiogenesis and describe a number of methodologies applicable to experimental manipulation of the retinal vascular system.     

Conversion of viable but nonculturable enteric bacteria to culturable by co-culture with eukaryotic cells

Viable but nonculturable (VBNC) Vibrio cholerae non-O1/non-O139, V. parahaemolyticus, enterohemorrhagic Escherichia coli, enterotoxigenic E. coli, enteropathogenic E. coli, Shigella flexneri, and Salmonella enterica were converted to the culturable state by co-culture with selected eukaryotic cells, e.g., HT-29, Caco-2, T84, HeLa, Intestine 407, and CHO cells.     

Realignment of signal processing within a sensory brainstem nucleus as brain temperature declines in the Syrian hamster, a hibernating species

Crucial for survival, the central nervous system must reliably process sensory information over all stages of a hibernation bout to ensure homeostatic regulation is maintained and well-matched to dramatically altered behavioral states. Comparing neural responses in the nucleus tractus solitarius of rats and euthermic Syrian hamsters, we tested the hypothesis that hamster neurons have adaptations sustaining signal processing while conserving energy. Using patch-clamp techniques, we classified second-order neurons in the nucleus as rapid-onset or delayed-onset spiking phenotypes based on their spiking onset to a depolarizing pulse (following a −80 mV prepulse). As temperature decreased from 33 to 15°C, the excitability of all neurons decreased. However, hamster rapid-onset spiking neurons had the highest spiking response and shortest action potential width at every temperature, while hamster delayed-onset spiking neurons had the most negative resting membrane potential. The frequency of spontaneous excitatory postsynaptic currents in both phenotypes decreased as temperature decreased, yet the amplitudes of tractus solitarius stimulation-evoked currents were greater in hamsters than in rats regardless of phenotype and temperature. Changes were significant (P < 0.05), supporting our hypothesis by showing that, as temperature falls, rapid-onset neurons contribute more to signal processing but less to energy conservation than do delayed-onset neurons.     

Spatial and temporal expressions of prune reveal a role in Müller gliogenesis during Xenopus retinal development

The development of stratified retinal cell architecture is highly conserved in all vertebrates, implying that a common fundamental molecular mechanism is involved in the generation of the organized retina. However, the detailed molecular mechanisms of retinal development are not fully understood. Here we have identified the Xenopus ortholog of prune and show that it is expressed in both differentiating and differentiated retinal domains during development. Interestingly, these spatial and temporal expression patterns coincide with the expression of prune binding partners, the NM23 family members. Overexpression of prune in retinal precursor cells significantly increases the ratio of Müller glial cells as observed by modulation of NM23 activity (Mochizuki et al., 2009). However, a mutated form of prune that has replacement of four aspartate (D) residues (D'Angelo et al., 2004), essential for phosphodiesterase activity, does not exhibit gliogenic activity. Our observations suggest that Xenopus prune may regulate Müller gliogenesis through phosphodiesterase-mediated regulation of NM23 family members.     

Imiquimod Suppresses Propagation of Herpes Simplex Virus 1 by Upregulation of Cystatin A via the Adenosine Receptor A1 Pathway

Imiquimod is recognized as an agonist for Toll-like receptor 7 (TLR7) in immunocompetent cells. TLR7, as well as TLR3 and TLR8, triggers the immune responses, such as the production of type I interferons (IFNs) and proinflammatory cytokines via recognition of viral nucleic acids in the infected cells. In this study, we proposed that imiquimod has an IFN-independent antiviral effect in nonimmune cells. Imiquimod, but not resiquimod, suppressed replication of human herpes simplex virus 1 (HSV-1) in FL cells. We analyzed alternation of gene expression by treatment with imiquimod using microarray analysis. Neither type I IFNs, nor TLRs, nor IFN-inducible antiviral genes were induced in imiquimod-treated FL cells. Cystatin A, a host cysteine protease inhibitor, was strongly upregulated by imiquimod and took a major part in the anti-HSV-1 activity deduced by the suppression experiment using its small interfering RNA. Upregulation of cystatin A was suggested to be mediated by antagonizing adenosine receptor A(1) and activating the protein kinase A pathway. Imiquimod, but not resiquimod, was shown to interact with adenosine receptor A(1). Imiquimod-induced anti-HSV-1 activity was observed in other cells, such as HeLa, SiHa, and CaSki cells, in a manner consistent with the cystatin A induction by imiquimod. These results indicated that imiquimod acted as an antagonist for adenosine receptor A(1) and induced a host antiviral protein, cystatin A. The process occurred independently of TLR7 and type I IFNs.     

Identification of genes involved in the acetamidino group modification of the flagellin N-linked glycan of Methanococcus maripaludis

N-linked glycosylation of protein is a posttranslational modification found in all three domains of life. The flagellin proteins of the archaeon Methanococcus maripaludis are known to be modified with an N-linked tetrasaccharide consisting of N-acetylgalactosamine (GalNAc), a diacetylated glucuronic acid (GlcNAc3NAc), an acetylated and acetamidino-modified mannuronic acid with a substituted threonine group (ManNAc3NAmA6Thr), and a novel terminal sugar residue [(5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose]. To identify genes involved in biosynthesis of the component sugars of this glycan, three genes, mmp1081, mmp1082, and mmp1083, were targeted for in-frame deletion, based on their annotation and proximity to glycosyltransferase genes known to be involved in assembly of the glycan. Mutants carrying a deletion in any of these three genes remained flagellated and motile. A strain with a deletion of mmp1081 had lower-molecular-mass flagellins in Western blots. Mass spectrometry of purified flagella revealed a truncated glycan with the terminal sugar absent and the threonine residue and the acetamidino group missing from the third sugar. No glycan modification was seen in either the Δmmp1082 or Δmmp1083 mutant grown in complex Balch III medium. However, a glycan identical to the Δmmp1081 glycan was observed when the Δmmp1082 or Δmmp1083 mutant was grown under ammonia-limited conditions. We hypothesize that MMP1082 generates ammonia and tunnels it through MMP1083 to MMP1081, which acts as the amidotransferase, modifying the third sugar residue of the M. maripaludis glycan with the acetamidino group.     

NMR and CD analysis of an intermediate state in the thermal unfolding process of mouse lipocalin-type prostaglandin D synthase

We previously reported that the thermal unfolding of mouse lipocalin-type prostaglandin D synthase (L-PGDS) is a completely reversible process under acidic conditions and follows a three-state pathway, including an intermediate state (I) between native state (N) and unfolded state. In the present study, we investigated the intermediate state of mouse C65A L-PGDS and clarified the local conformational changes in the upper and bottom regions by using NMR and CD spectroscopy. The (1)H-(15)N HSQC measurements revealed that the backbone conformation was disrupted in the upper region of the β-barrel at 45°C, which is around the T(m) value for the N ? I transition, but that the signals of the residues located at the bottom region of L-PGDS remained at 54°C, where the maximum accumulation of the intermediate state was found. (1)H-NMR and CD measurements showed that the T(m) values obtained by monitoring Trp54 at the upper region and Trp43 at the bottom region of the β-barrel were 41.4 and 47.5°C, respectively, suggesting that the conformational change in the upper region occurred at a lower temperature than that in the bottom region. These findings demonstrate that the backbone conformation of the bottom region is still maintained in the intermediate state.     

Up-regulation of blood arachidonate (20:4) levels in patients with chronic obstructive pulmonary disease

Context and objective: Plasma arachidonate (20:4) levels in patients with chronic obstructive pulmonary disease (COPD) were investigated. Methods: Plasma was extracted and free fatty acids (FFAs) were separated using column chromatography and measured by fluorescence. Plasma 20:4 levels and its percentage relative to total FFA levels (%20:4) were measured in COPD (n = 18) and control (n = 20) subjects. Results and conclusions: FFA levels were lower in COPD compared with normals. However, there was a significant increase in %20:4 levels in COPD patients (GOLD stage I/II 0.9 ± 0.4%; GOLD stage III/IV 1.1 ± 0.1%) compared with control subjects (0.6 ± 0.1, p < 0.05). %20:4 is a potential biomarker for COPD.     

Prevalence and Clinical Features of Hearing Loss Patients with CDH23 Mutations: A Large Cohort Study

Screening for gene mutations in CDH23, which has many exons, has lagged even though it is likely to be an important cause for hearing loss patients. To assess the importance of CDH23 mutations in non-syndromic hearing loss, two-step screening was applied and clinical characteristics of the patients with CDH23 mutations were examined in this study. As a first screening, we performed Sanger sequencing using 304 probands compatible with recessive inheritance to find the pathologic mutations. Twenty-six possible mutations were detected to be pathologic in the first screening. For the second screening, using the probes for these 26 mutations, a large cohort of probands (n = 1396) was screened using Taqman amplification-based mutation analysis followed by Sanger sequencing. The hearing loss in a total of 52 families (10 homozygous, 13 compound heterogygous, and 29 heterozygous) was found to be caused by the CDH23 mutations. The majority of the patients showed congenital, high frequency involved, progressive hearing loss. Interestingly, some particular mutations cause late onset moderate hearing loss. The present study is the first to demonstrate the prevalence of CDH23 mutations among non-syndromic hearing loss patients and indicated that mutations of the CDH23 gene are an important cause of non-syndromic hearing loss.