Sphingolipids in ocular inflammation.

Sphingolipids are essential to cell membrane structure and the development and maintenance of neural tissues. The role of bioactive sphingolipids has been established in numerous cellular events, including cell survival, growth, and apoptosis. Ocular inflammatory and autoimmune diseases involve activation and migration of endothelial cells, neovascularization, and infiltration of immune cells into various tissues. Clinically, the impact and role of sphingolipid-mediated signaling is increasingly being appreciated in the pathogenesis and treatment of diseases ranging from multiple sclerosis to neovascularization in age-related macular degeneration and diabetic retinopathy. In this review, we discuss our current knowledge and understanding of sphingolipid metabolism and signaling associated with the pathogenesis of ocular diseases.

Ocular injuries from shake and bake methamphetamine labs.

PURPOSE: To review ocular injuries resulting from "shake and bake" methamphetamine labs. METHOD: Retrospective case series of 4 patients with ocular injuries sustained from "shake and bake" lab explosions. RESULTS: Four men ages 20-39 underwent complete ophthalmologic examination after an injury from a "shake and bake" methamphetamine lab explosion.The mechanism of injury was initially misrepresented in each case; the physical findings were suggestive of thermal and alkali injury. Visual acuity ranged widely from 20/20 to light perception only. Treatment in the acute setting included irrigation, pH monitoring, and intraocular pressure lowering. CONCLUSION: Methamphetamine production by means of the"shake and bake" method can result in combined thermal and alkali ocular injury. Patients who sustain this type of injury may not accurately report the mechanism of exposure. Increased awareness of this type of ocular injury may increase the rapidity of diagnosis, avoid early misdiagnosis, and ultimately improve outcomes.

Interaction of a putative transcriptional regulatory protein and the thermo-inducible cts-52 mutant repressor in the Bacillus subtilis phage phi105 genome.

A 144 amino acid residue cts-52 mutant repressor (mtc phi 105) located in the EcoRI-F immunity region (immF) of Bacillus subtilis phage phi 105 is involved in the control mechanism of a thermo-inducible expression system. Adjacent to the repressor gene, an open-reading frame, designated ORF4, encodes a polypeptide of 90 amino acid residues, which shares a 37% homology with the amino acid sequence of the repressor. On the basis of the protein sequence alignment, a DNA-binding alpha helix-beta turn-alpha helix (HTH) motif was identified in the N-terminal region (residues 18-37) of the repressor as well as in the polypeptide of ORF4 (residues 22-41). In vivo expression of the mutant repressor and ORF4 were confirmed by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and Western blot analysis. To study their DNA binding properties, the wild-type repressor (wtc phi 105) and the mutant repressor mtc phi 105, which has a Thr17 to Ile substitution, were overexpressed in Escherichia coli and purified for affinity assays. Their affinities towards six operator sites at various temperatures were elucidated by surface plasmon resonance (SPR). Our data showed that a temperature shift does not influence the wtc phi 105-operators' binding affinity, while the binding of mtc phi 105 to the operators was temperature sensitive. This explains how thermo-induction triggers the release of the mutant repressor and renders heterologous gene expression. Interestingly, mtc phi 105 and ORF4 demonstrated a large affinity discrepancy towards individual operators at different temperatures. mRNA levels monitored by real-time RT-PCR indicated a suppression of mtc phi 105 expression, but a stimulation of ORF4 transcription after thermo-induction. Our data suggested that ORF4 might be a counter protein to the phage repressor in the modulation of the two divergent-oriented promoters P(M) and P(R) within the immF region.

Factors associated with age of onset of herpes zoster ophthalmicus.

PURPOSE: The aim of this study was to evaluate factors associated with the age of onset of herpes zoster ophthalmicus (HZO) in the era of varicella vaccination. METHODS: A retrospective series of 400 subjects with a diagnosis of HZO, and an analysis of public health databases. Variables studied included the age at onset, period of disease onset (1996-2004 and 2005-2012), sex, smoking habits, diabetes status, autoimmunity, and immunosuppressed status. Community data were gathered from the Oklahoma Outpatient Surgery Discharge Public Use Data File and the United States Census' American Community Survey data set. RESULTS: The mean onset age was significantly lower for 2005 to 2012 as compared with 1996 to 2004 among females (mean decrease, 9.3 years; 95% confidence interval, 4.6-13.9 years; P < 0.0001), but the mean onset age was similar between the 2 periods among males (P = 0.640). Whereas 32.3% of the patients with zoster were <60 years old in 1996 to 2004, compared with 44.8% in the 2005 to 2012 period (χ test: P = 0.017). In the multivariate model, smokers were found to have disease onset 11.5 (95% confidence interval, 6.9-16.1) years younger than nonsmokers. CONCLUSIONS: The proportion of younger patients with HZO increased, whereas the institutional and community data sets demonstrate a downward shift of the average age of onset of HZO among females. This may be an effect of widespread childhood varicella vaccination. Immunosuppression and smoking were also associated with a younger age of onset of HZO. This has implications for clinical care of patients at risk for developing HZO, as well as public health and vaccination policies.

Transgenic plant-derived siRNAs can suppress propagation of influenza virus in mammalian cells.

As an example of the cost-effective large-scale generation of small-interfering RNA (siRNAs), we have created transgenic tobacco plants that produce siRNAs targeted to the mRNA of the non-structural protein NS1 from the influenza A virus subtype H1N1. We have investigated if these siRNAs, specifically targeted to the 5'-portion of the NS1 transcripts (5mNS1), would suppress viral propagation in mammalian cells. Agroinfiltration of transgenic tobacco with an Agrobacterium strain harboring a 5mNS1-expressing binary vector caused a reduction in 5mNS1 transcripts in the siRNA-accumulating transgenic plants. Further, H1N1 infection of siRNA-transfected mammalian cells resulted in significant suppression of viral replication. These results demonstrate that plant-derived siRNAs can inhibit viral propagation through RNA interference and could potentially be applied in control of viral-borne diseases.

Beyond the cherry-red spot: Ocular manifestations of sphingolipid-mediated neurodegenerative and inflammatory disorders.

Sphingolipids are a ubiquitous membrane lipid present in every cell and found most abundantly in neural tissues. Disorders such as Tay-Sachs or Niemann-Pick disease are the most familiar examples of dysfunction in sphingolipid metabolism and are typically associated with neurodegeneration and ocular findings such as blindness. More recently, the role of bioactive sphingolipids has been established in a multitude of cellular events, including cell survival, growth, senescence and apoptosis, inflammation, and neovascularization. We discuss our current knowledge and understanding of sphingolipid metabolism and signaling in the pathogenesis of ocular diseases.

Mechanisms and functional implications of the degradation of host RNA polymerase II in influenza virus infected cells.

Influenza viruses induce a host shut off mechanism leading to the general inhibition of host gene expression in infected cells. Here, we report that the large subunit of host RNA polymerase II (Pol II) is degraded in infected cells and propose that this degradation is mediated by the viral RNA polymerase that associates with Pol II. We detect increased ubiquitylation of Pol II in infected cells and upon the expression of the viral RNA polymerase suggesting that the proteasome pathway plays a role in Pol II degradation. Furthermore, we find that expression of the viral RNA polymerase results in the inhibition of Pol II transcription. We propose that Pol II inhibition and degradation in influenza virus infected cells could represent a viral strategy to evade host antiviral defense mechanisms. Our results also suggest a mechanism for the temporal regulation of viral mRNA synthesis.

Influenza virus inhibits RNA polymerase II elongation.

The influenza virus RNA-dependent RNA polymerase interacts with the serine-5 phosphorylated carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II). It was proposed that this interaction allows the viral RNA polymerase to gain access to host mRNA-derived capped RNA fragments required as primers for the initiation of viral mRNA synthesis. Here, we show, using a chromatin immunoprecipitation (ChIP) analysis, that similar amounts of Pol II associate with Pol II promoter DNAs in influenza virus-infected and mock-infected cells. However, there is a statistically significant reduction in Pol II densities in the coding region of Pol II genes in infected cells. Thus, influenza virus specifically interferes with Pol II elongation, but not Pol II initiation. We propose that influenza virus RNA polymerase, by binding to the CTD of initiating Pol II and subsequent cleavage of the capped 5' end of the nascent transcript, triggers premature Pol II termination.

A dual protein expression system in Bacillus subtilis.

We have developed a dual expression system for the simultaneous overexpression of two proteins in Bacillus subtilis. Two candidate genes, xylanase (xynA) and glucanase (bglS) from B. subtilis strain 168, which were engineered with independent Shine-Dalgarno (SD) sequences, were cloned in tandem into a transfer vector, which was then transformed into B. subtilis strain 1A304 (phi105MU331). The genes were under the transcriptional control of a strong promoter of a bacteriophage, phi105, where transcription was initiated upon thermal induction. Six constructs were made to compare the factors that affected the yields of the gene products. The expression level of each candidate gene was found to correspond to its position relative to the phage promoter, irrespective of the identity of the insert. The lower expression level of the second insert might have been due to limited resources for protein synthesis, a short half-life of the mRNA, or an early termination of the RNA polymerase. Curiously, gene duplications in tandem did not lead to further increase in production.

The bi-directional translocation of MARCKS between membrane and cytosol regulates integrin-mediated muscle cell spreading.

The regulation of the cytoskeleton is critical to normal cell function during tissue morphogenesis. Cell-matrix interactions mediated by integrins regulate cytoskeletal dynamics, but the signaling cascades that control these processes remain largely unknown. Here we show that myristoylated alanine-rich C-kinase substrate (MARCKS) a specific substrate of protein kinase C (PKC), is regulated by alpha5beta1 integrin-mediated activation of PKC and is critical to the regulation of actin stress fiber formation during muscle cell spreading. Using MARCKS mutants that are defective in membrane association or responsiveness to PKC-dependent phosphorylation, we demonstrate that the translocation of MARCKS from the membrane to the cytosol in a PKC-dependent manner permits the initial phases of cell adhesion. The dephosphorylation of MARCKS and its translocation back to the membrane permits the later stages of cell spreading during the polymerization and cross-linking of actin and the maturation of the cytoskeleton. All of these processes are directly dependent on the binding of alpha5beta1 integrin to its extracellular matrix receptor, fibronectin. These results demonstrate a direct biochemical pathway linking alpha5beta1 integrin signaling to cytoskeletal dynamics and involving bi-directional translocation of MARCKS during the dramatic changes in cellular morphology that occur during cell migration and tissue morphogenesis.