[Storage Stability of Nitrite in Nitrite Analysis of Meat Products].

For the analysis of nitrite ions in food, the stabilities of nitrite ions in meat products and their standard solutions were evaluated. Nitrite is easily oxidized or reduced; hence, products with standard solutions or colour retention agent must be carefully handled. To assess the stability and decreasing trend of nitrite, we examined the storage stability of standard solutions using calibration curves, the time course of nitrite in chopped meat products stored under different conditions, and the time course of nitrite in the sample solutions. Regarding calibration curves, the storage stability was determined for standard solutions that were prepared with ultrapure water at concentrations of 0.025 and 0.4 µg/mL and were stored at 5℃ for one year. The results revealed no changes in concentration of any solution over time, suggesting that no readjustments to the standard solution concentration were necessary before testing until one year after their preparation. Time course of nitrite in chopped meat products stored under different conditions showed a significant decrease in nitrite in refrigerated storage (5℃), whereas stability of nitrite was maintained for up to 1 day in frozen storage (-20℃) and for 14 days in frozen storage (-40℃). The time course of nitrite in the sample solutions showed that the quantitative values of nitrite in the extract remained unchanged within one week of extraction for the meat products tested in the study.

Genetic and Functional Analyses of Archaeal ATP-Dependent RNA Ligase in C/D Box sRNA Circularization and Ribosomal RNA Processing.

RNA ligases play important roles in repairing and circularizing RNAs post-transcriptionally. In this study, we generated an allelic knockout of ATP-dependent RNA ligase (Rnl) in the hyperthermophilic archaeon Thermococcus kodakarensis to identify its biological targets. A comparative analysis of circular RNA reveals that the Rnl-knockout strain represses circularization of C/D box sRNAs without affecting the circularization of tRNA and rRNA processing intermediates. Recombinant archaeal Rnl could circularize C/D box sRNAs with a mutation in the conserved C/D box sequence element but not when the terminal stem structures were disrupted, suggesting that proximity of the two ends could be critical for intramolecular ligation. Furthermore, T. kodakarensis accumulates aberrant RNA fragments derived from ribosomal RNA in the absence of Rnl. These results suggest that Rnl is responsible for C/D box sRNA circularization and may also play a role in ribosomal RNA processing.

[Study of Rapid HPLC Method for the Determination of Antioxidants in Foods, and Standard Solution Stability during Storage].

In this study, we simultaneously determined three antioxidants, butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA), and tert-butylhydroquinone (TBHQ), using HPLC equipped both with a photodiode array detector and a fluorescence detector in 25 minutes per sample. Due to the combined use of the two detectors, we could achieve improved target selectivity. Further, quantification at the specific wavelengths for each target substance particularly increased BHT detection sensitivity. This approach enabled us to avoid repeated measurements during daily inspections. Furthermore, detections were performed using LC-MS/MS instead of GC-MS to overcome the problem of helium gas shortage.In addition, we investigated antioxidant stability in standard solutions during storage. Although TBHQ was stable in methanol with ascorbic acid at -20℃, ascorbic acid storage has possibility to lead to decrease in BHT and BHA concentrations. We recognized that the mixture of BHT and BHA dissolved in methanol at 4℃ and that of BHT, BHA and TBHQ dissolved in methanol with ascorbic acid at -20℃ were suitable for about one year.

Differentiating Trypanosoma cruzi in a Host Mammalian Cell Imaged in Aqueous Liquid by Atmospheric Scanning Electron Microscopy.

Atmospheric Scanning Electron Microscopy (ASEM) is a powerful tool to observe a wet specimen at high resolution under atmospheric pressure. Here, we visualized a protozoan parasite Trypanosoma cruzi over the course of its infection cycle in the host mammalian cell. This is the first observation of intracellular parasite using a liquid-phase EM. Unlike regular SEM, aldehyde-fixed cell body of T. cruzi appears translucent, allowing the visualization of internal structures such as kinetoplast of trypomastigote and nucleus of amastigote. Plasma membrane of the host mammalian cell also appears translucent, which enabled direct observation of differentiating intracellular parasites and dynamic change of host cellular structures in their near-natural states. Various water-rich structures including micro- and macro- vesicles were visualized around T. cruzi. In addition, Correlative Light and Electron Microscopy exploiting open sample dish of ASEM allowed identification of parasite nucleus and transfected fluorescence-labeled parasites soon after internalization, while location of this morphological intermediate was otherwise obscure. Successful visualization of the differentiation of T. cruzi within the host cell demonstrated here opens up the possibility of using ASEM for observation of variety of intracellular parasites. IMPORTANCE Using Atmospheric Scanning Electron Microscopy (ASEM), we visualized interaction between infectious stage of Trypanosoma cruzi and completely intact host mammalian cell. Plasma membrane appears translucent under ASEM, which not only enables direct observation of T. cruzi within its host cell, but also reveals internal structures of the parasite itself. Sample deformation is minimal, since the specimen remains hydrated under atmospheric pressure at all times. This nature of ASEM, along with the open structure of ASEM sample dish, is suited for correlative light-electron microscopy, which can further be exploited in identification of fluorescent protein in the intracellular parasites.

Crystal structures of the RNA triphosphatase from Trypanosoma cruzi provide insights into how it recognizes the 5'-end of the RNA substrate.

RNA triphosphatase catalyzes the first step in mRNA cap formation, hydrolysis of the terminal phosphate from the nascent mRNA transcript. The RNA triphosphatase from the protozoan parasite Trypanosoma cruzi, TcCet1, belongs to the family of triphosphate tunnel metalloenzymes (TTMs). TcCet1 is a promising antiprotozoal drug target because the mechanism and structure of the protozoan RNA triphosphatases are completely different from those of the RNA triphosphatases found in mammalian and arthropod hosts. Here, we report several crystal structures of the catalytically active form of TcCet1 complexed with a divalent cation and an inorganic tripolyphosphate in the active-site tunnel at 2.20-2.51 Å resolutions. The structures revealed that the overall structure, the architecture of the tunnel, and the arrangement of the metal-binding site in TcCet1 are similar to those in other TTM proteins. On the basis of the position of three sulfate ions that cocrystallized on the positively charged surface of the protein and results obtained from mutational analysis, we identified an RNA-binding site in TcCet1. We conclude that the 5'-end of the triphosphate RNA substrate enters the active-site tunnel directionally. The structural information reported here provides valuable insight into designing inhibitors that could specifically block the entry of the triphosphate RNA substrate into the TTM-type RNA triphosphatases of T. cruzi and related pathogens.

Introducing a Gene Knockout Directly Into the Amastigote Stage of Trypanosoma cruzi Using the CRISPR/Cas9 System.

Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas' disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.

Utilization of proliferable extracellular amastigotes for transient gene expression, drug sensitivity assay, and CRISPR/Cas9-mediated gene knockout in Trypanosoma cruzi.

Trypanosoma cruzi has three distinct life cycle stages; epimastigote, trypomastigote, and amastigote. Amastigote is the replication stage in host mammalian cells, hence this stage of parasite has clinical significance in drug development research. Presence of extracellular amastigotes (EA) and their infection capability have been known for some decades. Here, we demonstrate that EA can be utilized as an axenic culture to aid in stage-specific study of T. cruzi. Amastigote-like property of axenic amastigote can be sustained in LIT medium at 37°C at least for 1 week, judging from their morphology, amastigote-specific UTR-regulated GFP expression, and stage-specific expression of selected endogenous genes. Inhibitory effect of benznidazole and nifurtimox on axenic amastigotes was comparable to that on intracellular amastigotes. Exogenous nucleic acids can be transfected into EA via conventional electroporation, and selective marker could be utilized for enrichment of transfectants. We also demonstrate that CRISPR/Cas9-mediated gene knockout can be performed in EA. Essentiality of the target gene can be evaluated by the growth capability of the knockout EA, either by continuation of axenic culturing or by host infection and following replication as intracellular amastigotes. By taking advantage of the accessibility and sturdiness of EA, we can potentially expand our experimental freedom in studying amastigote stage of T. cruzi.

Development of a motion-based cell-counting system for Trypanosoma parasite using a pattern recognition approach.

Automated cell counters that utilize still images of sample cells are widely used. However, they are not well suited to counting slender, aggregate-prone microorganisms such as Trypanosoma cruzi. Here, we developed a motion-based cell-counting system, using an image-recognition method based on a cubic higher-order local auto-correlation feature. The software successfully estimated the cell density of dispersed, aggregated, as well as fluorescent parasites by motion pattern recognition. Loss of parasites activeness due to drug treatment could also be detected as a reduction in apparent cell count, which potentially increases the sensitivity of drug screening assays. Moreover, the motion-based approach enabled estimation of the number of parasites in a co-culture with host mammalian cells, by disregarding the presence of the host cells as a static background.

Crystal structures of U6 snRNA-specific terminal uridylyltransferase.

The terminal uridylyltransferase, TUT1, builds or repairs the 3'-oligo-uridylylated tail of U6 snRNA. The 3'-oligo-uridylylated tail is the Lsm-binding site for U4/U6 di-snRNP formation and U6 snRNA recycling for pre-mRNA splicing. Here, we report crystallographic and biochemical analyses of human TUT1, which revealed the mechanisms for the specific uridylylation of the 3'-end of U6 snRNA by TUT1. The O2 and O4 atoms of the UTP base form hydrogen bonds with the conserved His and Asn in the catalytic pocket, respectively, and TUT1 preferentially incorporates UMP onto the 3'-end of RNAs. TUT1 recognizes the entire U6 snRNA molecule by its catalytic domains, N-terminal RNA-recognition motifs and a previously unidentified C-terminal RNA-binding domain. Each domain recognizes specific regions within U6 snRNA, and the recognition is coupled with the domain movements and U6 snRNA structural changes. Hence, TUT1 functions as the U6 snRNA-specific terminal uridylyltransferase required for pre-mRNA splicing.

Nanomolar Inhibitors of Trypanosoma brucei RNA Triphosphatase.

UNLABELLED: Eukaryal taxa differ with respect to the structure and mechanism of the RNA triphosphatase (RTPase) component of the mRNA capping apparatus. Protozoa, fungi, and certain DNA viruses have a metal-dependent RTPase that belongs to the triphosphate tunnel metalloenzyme (TTM) superfamily. Because the structures, active sites, and chemical mechanisms of the TTM-type RTPases differ from those of mammalian RTPases, the TTM RTPases are potential targets for antiprotozoal, antifungal, and antiviral drug discovery. Here, we employed RNA interference (RNAi) knockdown methods to show that Trypanosoma brucei RTPase Cet1 (TbCet1) is necessary for proliferation of procyclic cells in culture. We then conducted a high-throughput biochemical screen for small-molecule inhibitors of the phosphohydrolase activity of TbCet1. We identified several classes of chemicals-including chlorogenic acids, phenolic glycopyranosides, flavonoids, and other phenolics-that inhibit TbCet1 with nanomolar to low-micromolar 50% inhibitory concentrations (IC50s). We confirmed the activity of these compounds, and tested various analogs thereof, by direct manual assays of TbCet1 phosphohydrolase activity. The most potent nanomolar inhibitors included tetracaffeoylquinic acid, 5-galloylgalloylquinic acid, pentagalloylglucose, rosmarinic acid, and miquelianin. TbCet1 inhibitors were less active (or inactive) against the orthologous TTM-type RTPases of mimivirus, baculovirus, and budding yeast (Saccharomyces cerevisiae). Our results affirm that a TTM RTPase is subject to potent inhibition by small molecules, with the caveat that parallel screens against TTM RTPases from multiple different pathogens may be required to fully probe the chemical space of TTM inhibition. IMPORTANCE: The stark differences between the structure and mechanism of the RNA triphosphatase (RTPase) component of the mRNA capping apparatus in pathogenic protozoa, fungi, and viruses and those of their metazoan hosts highlight RTPase as a target for anti-infective drug discovery. Protozoan, fungal, and DNA virus RTPases belong to the triphosphate tunnel metalloenzyme family. This study shows that a protozoan RTPase, TbCet1 from Trypanosoma brucei, is essential for growth of the parasite in culture and identifies, via in vitro screening of chemical libraries, several classes of potent small-molecule inhibitors of TbCet1 phosphohydrolase activity.

Multitarget, quantitative nanoplasmonic electrical field-enhanced resonating device (NE2RD) for diagnostics.

Recent advances in biosensing technologies present great potential for medical diagnostics, thus improving clinical decisions. However, creating a label-free general sensing platform capable of detecting multiple biotargets in various clinical specimens over a wide dynamic range, without lengthy sample-processing steps, remains a considerable challenge. In practice, these barriers prevent broad applications in clinics and at patients' homes. Here, we demonstrate the nanoplasmonic electrical field-enhanced resonating device (NE(2)RD), which addresses all these impediments on a single platform. The NE(2)RD employs an immunodetection assay to capture biotargets, and precisely measures spectral color changes by their wavelength and extinction intensity shifts in nanoparticles without prior sample labeling or preprocessing. We present through multiple examples, a label-free, quantitative, portable, multitarget platform by rapidly detecting various protein biomarkers, drugs, protein allergens, bacteria, eukaryotic cells, and distinct viruses. The linear dynamic range of NE(2)RD is five orders of magnitude broader than ELISA, with a sensitivity down to 400 fg/mL This range and sensitivity are achieved by self-assembling gold nanoparticles to generate hot spots on a 3D-oriented substrate for ultrasensitive measurements. We demonstrate that this precise platform handles multiple clinical samples such as whole blood, serum, and saliva without sample preprocessing under diverse conditions of temperature, pH, and ionic strength. The NE(2)RD's broad dynamic range, detection limit, and portability integrated with a disposable fluidic chip have broad applications, potentially enabling the transition toward precision medicine at the point-of-care or primary care settings and at patients' homes.

The messenger RNA decapping and recapping pathway in Trypanosoma.

The 5' terminus of trypanosome mRNA is protected by a hypermethylated cap 4 derived from spliced leader (SL) RNA. Trypanosoma brucei nuclear capping enzyme with cap guanylyltransferase and methyltransferase activities (TbCgm1) modifies the 5'-diphosphate RNA (ppRNA) end to generate an m7G SL RNA cap. Here we show that T. brucei cytoplasmic capping enzyme (TbCe1) is a bifunctional 5'-RNA kinase and guanylyltransferase that transfers a γ-phosphate from ATP to pRNA to form ppRNA, which is then capped by transfer of GMP from GTP to the RNA ß-phosphate. A Walker A-box motif in the N-terminal domain is essential for the RNA kinase activity and is targeted preferentially to a SL RNA sequence with a 5'-terminal methylated nucleoside. Silencing of TbCe1 leads to accumulation of uncapped mRNAs, consistent with selective capping of mRNA that has undergone trans-splicing and decapping. We identify T. brucei mRNA decapping enzyme (TbDcp2) that cleaves m7GDP from capped RNA to generate pRNA, a substrate for TbCe1. TbDcp2 can also remove GDP from unmethylated capped RNA but is less active at a mature cap 4 end and thus may function in RNA cap quality surveillance. Our results establish the enzymology and relevant protein catalysts of a cytoplasmic recapping pathway that has broad implications for the functional reactivation of processed mRNA ends.

Three-dimensional structure of a protozoal double-stranded RNA virus that infects the enteric pathogen Giardia lamblia.

UNLABELLED: Giardia lamblia virus (GLV) is a small, nonenveloped, nonsegmented double-stranded RNA (dsRNA) virus infecting Giardia lamblia, the most common protozoan pathogen of the human intestine and a major agent of waterborne diarrheal disease worldwide. GLV (genus Giardiavirus) is a member of family Totiviridae, along with several other groups of protozoal or fungal viruses, including Leishmania RNA viruses and Trichomonas vaginalis viruses. Interestingly, GLV is more closely related than other Totiviridae members to a group of recently discovered metazoan viruses that includes penaeid shrimp infectious myonecrosis virus (IMNV). Moreover, GLV is the only known protozoal dsRNA virus that can transmit efficiently by extracellular means, also like IMNV. In this study, we used transmission electron cryomicroscopy and icosahedral image reconstruction to examine the GLV virion at an estimated resolution of 6.0 Å. Its outermost diameter is 485 Å, making it the largest totivirus capsid analyzed to date. Structural comparisons of GLV and other totiviruses highlighted a related "T=2" capsid organization and a conserved helix-rich fold in the capsid subunits. In agreement with its unique capacity as a protozoal dsRNA virus to survive and transmit through extracellular environments, GLV was found to be more thermoresistant than Trichomonas vaginalis virus 1, but no specific protein machinery to mediate cell entry, such as the fiber complexes in IMNV, could be localized. These and other structural and biochemical findings provide a basis for future work to dissect the cell entry mechanism of GLV into a "primitive" (early-branching) eukaryotic host and an important enteric pathogen of humans. IMPORTANCE: Numerous pathogenic bacteria, including Corynebacterium diphtheriae, Salmonella enterica, and Vibrio cholerae, are infected with lysogenic bacteriophages that contribute significantly to bacterial virulence. In line with this phenomenon, several pathogenic protozoa, including Giardia lamblia, Leishmania species, and Trichomonas vaginalis are persistently infected with dsRNA viruses, and growing evidence indicates that at least some of these protozoal viruses can likewise enhance the pathogenicity of their hosts. Understanding of these protozoal viruses, however, lags far behind that of many bacteriophages. Here, we investigated the dsRNA virus that infects the widespread enteric parasite Giardia lamblia. Using electron cryomicroscopy and icosahedral image reconstruction, we determined the virion structure of Giardia lamblia virus, obtaining new information relating to its assembly, stability, functions in cell entry and transcription, and similarities and differences with other dsRNA viruses. The results of our study set the stage for further mechanistic work on the roles of these viruses in protozoal virulence.

The villain team-up or how Trichomonas vaginalis and bacterial vaginosis alter innate immunity in concert.

OBJECTIVES: Complex interactions of vaginal microorganisms with the genital tract epithelium shape mucosal innate immunity, which holds the key to sexual and reproductive health. Bacterial vaginosis (BV), a microbiome-disturbance syndrome prevalent in reproductive-age women, occurs commonly in concert with trichomoniasis, and both are associated with increased risk of adverse reproductive outcomes and viral infections, largely attributable to inflammation. To investigate the causative relationships among inflammation, BV and trichomoniasis, we established a model of human cervicovaginal epithelial cells colonised by vaginal Lactobacillus isolates, dominant in healthy women, and common BV species (Atopobium vaginae, Gardnerella vaginalis and Prevotella bivia). METHODS: Colonised epithelia were infected with Trichomonas vaginalis (TV) or exposed to purified TV virulence factors (membrane lipophosphoglycan (LPG), its ceramide-phosphoinositol-glycan core (CPI-GC) or the endosymbiont Trichomonas vaginalis virus (TVV)), followed by assessment of bacterial colony-forming units, the mucosal anti-inflammatory microbicide secretory leucocyte protease inhibitor (SLPI), and chemokines that drive pro-inflammatory, antigen-presenting and T cells. RESULTS: TV reduced colonisation by Lactobacillus but not by BV species, which were found inside epithelial cells. TV increased interleukin (IL)-8 and suppressed SLPI, likely via LPG/CPI-GC, and upregulated IL-8 and RANTES, likely via TVV as suggested by use of purified pathogenic determinants. BV species A vaginae and G vaginalis induced IL-8 and RANTES, and also amplified the pro-inflammatory responses to both LPG/CPI-GC and TVV, whereas P bivia suppressed the TV/TVV-induced chemokines. CONCLUSIONS: These molecular host-parasite-endosymbiont-bacteria interactions explain epidemiological associations and suggest a revised paradigm for restoring vaginal immunity and preventing BV/TV-attributable inflammatory sequelae in women.

Structure of a protozoan virus from the human genitourinary parasite Trichomonas vaginalis.

The flagellated protozoan Trichomonas vaginalis is an obligate human genitourinary parasite and the most frequent cause of sexually transmitted disease worldwide. Most clinical isolates of T. vaginalis are persistently infected with one or more double-stranded RNA (dsRNA) viruses from the genus Trichomonasvirus, family Totiviridae, which appear to influence not only protozoan biology but also human disease. Here we describe the three-dimensional structure of Trichomonas vaginalis virus 1 (TVV1) virions, as determined by electron cryomicroscopy and icosahedral image reconstruction. The structure reveals a T = 1 capsid comprising 120 subunits, 60 in each of two nonequivalent positions, designated A and B, as previously observed for fungal Totiviridae family members. The putative protomer is identified as an asymmetric AB dimer consistent with either decamer or tetramer assembly intermediates. The capsid surface is notable for raised plateaus around the icosahedral 5-fold axes, with canyons connecting the 2- and 3-fold axes. Capsid-spanning channels at the 5-fold axes are unusually wide and may facilitate release of the viral genome, promoting dsRNA-dependent immunoinflammatory responses, as recently shown upon the exposure of human cervicovaginal epithelial cells to either TVV-infected T. vaginalis or purified TVV1 virions. Despite extensive sequence divergence, conservative features of the capsid reveal a helix-rich fold probably derived from an ancestor shared with fungal Totiviridae family members. Also notable are mass spectrometry results assessing the virion proteins as a complement to structure determination, which suggest that translation of the TVV1 RNA-dependent RNA polymerase in fusion with its capsid protein involves -2, and not +1, ribosomal frameshifting, an uncommonly found mechanism to date.

Fibers come and go: differences in cell-entry components among related dsRNA viruses.

Encapsidated dsRNA viruses, most of which are nonenveloped, infect a wide variety of hosts, from bacteria to vertebrates, and are currently grouped into 9 families comprising 33 genera. Given this range, it is not surprising that substantial diversity is seen in their transmission strategies and cell-entry machineries. One interesting set of recent findings is that several of these viruses, otherwise closely related, exhibit major differences in their entry machineries without comparably major differences in their capsid organizations. Examples are presence or absence of receptor-binding fibers among orthoreoviruses and aquareoviruses and presence or absence of both binding and membrane-penetration modules among totiviruses and between picobirnaviruses and partitiviruses. Evolved differences in cell-entry components among these viruses are therefore not only common but also seemingly straightforward from a structural standpoint.

Endobiont viruses sensed by the human host - beyond conventional antiparasitic therapy.

Wide-spread protozoan parasites carry endosymbiotic dsRNA viruses with uncharted implications to the human host. Among them, Trichomonas vaginalis, a parasite adapted to the human genitourinary tract, infects globally ∼250 million each year rendering them more susceptible to devastating pregnancy complications (especially preterm birth), HIV infection and HPV-related cancer. While first-line antibiotic treatment (metronidazole) commonly kills the protozoan pathogen, it fails to improve reproductive outcome. We show that endosymbiotic Trichomonasvirus, highly prevalent in T. vaginalis clinical isolates, is sensed by the human epithelial cells via Toll-like receptor 3, triggering Interferon Regulating Factor -3, interferon type I and proinflammatory cascades previously implicated in preterm birth and HIV-1 susceptibility. Metronidazole treatment amplified these proinflammatory responses. Thus, a new paradigm targeting the protozoan viruses along with the protozoan host may prevent trichomoniasis-attributable inflammatory sequelae.

The effects of light regimes and hormones on corneal growth in vivo and in organ culture.

When chicks are exposed to constant light (CL) during growth, their corneas become flatter and lighter in weight, and their anterior segments become shallower than those of chicks exposed to cyclical periods of light and dark. These effects have been correlated with CL suppression of cyclical changes in melatonin levels. The question of whether light directly influences corneal growth (e.g. via cryptochromes in the cornea) or acts remotely via the suppression of the melatonin rhythm has not yet been answered. Retinoic acid (RA), an ubiquitous morphogen, also causes non-functional flattening during corneal growth, but its effect in vivo has not been correlated with light regimes. We wished to characterize and distinguish between hormonal and light effects on corneal growth. We used organ culture to study the direct effects of light regimes, melatonin, and RA, and compared these results with those of parallel in vivo experiments. In this study, eye drops containing melatonin or RA were applied to corneas exposed to CL in vivo or in organ culture, and effects on corneal mass and hydration were measured. We applied a melatonin blocker, luzindole, to chick corneas in normal light/dark conditions to confirm that the observed melatonin effects are mediated at the cell membrane. Anterior chamber depth and refraction in vivo were measured. We found that, during CL exposure, combined application of melatonin and RA eye drops increased the depth of the anterior segment in vivo, (P = 0.003) and interestingly, both also reduced the hyperopia of CL exposure after 2 weeks (P = 0.002), thus partially reversing the effects of CL. RA increased corneal hydration in vivo (P = 0.030) but not in organ culture. Melatonin had no effect on corneal hydration in vivo, but in organ culture, melatonin significantly decreased hydration (P < 0.001). We found no evidence for a direct effect of light on corneal hydration in growing chick corneas in culture. Melatonin is required for normal corneal growth in vivo, and together melatonin and RA, or RA alone, affects the regulation of water content within the chick cornea. Melatonin also affects corneal hydration in vitro, but RA does not.

Phospholipase D family member 4, a transmembrane glycoprotein with no phospholipase D activity, expression in spleen and early postnatal microglia.

BACKGROUND: Phospholipase D (PLD) catalyzes conversion of phosphatidylcholine into choline and phosphatidic acid, leading to a variety of intracellular signal transduction events. Two classical PLDs, PLD1 and PLD2, contain phosphatidylinositide-binding PX and PH domains and two conserved His-x-Lys-(x)(4)-Asp (HKD) motifs, which are critical for PLD activity. PLD4 officially belongs to the PLD family, because it possesses two HKD motifs. However, it lacks PX and PH domains and has a putative transmembrane domain instead. Nevertheless, little is known regarding expression, structure, and function of PLD4. METHODOLOGY/PRINCIPAL FINDINGS: PLD4 was analyzed in terms of expression, structure, and function. Expression was analyzed in developing mouse brains and non-neuronal tissues using microarray, in situ hybridization, immunohistochemistry, and immunocytochemistry. Structure was evaluated using bioinformatics analysis of protein domains, biochemical analyses of transmembrane property, and enzymatic deglycosylation. PLD activity was examined by choline release and transphosphatidylation assays. Results demonstrated low to modest, but characteristic, PLD4 mRNA expression in a subset of cells preferentially localized around white matter regions, including the corpus callosum and cerebellar white matter, during the first postnatal week. These PLD4 mRNA-expressing cells were identified as Iba1-positive microglia. In non-neuronal tissues, PLD4 mRNA expression was widespread, but predominantly distributed in the spleen. Intense PLD4 expression was detected around the marginal zone of the splenic red pulp, and splenic PLD4 protein recovered from subcellular membrane fractions was highly N-glycosylated. PLD4 was heterologously expressed in cell lines and localized in the endoplasmic reticulum and Golgi apparatus. Moreover, heterologously expressed PLD4 proteins did not exhibit PLD enzymatic activity. CONCLUSIONS/SIGNIFICANCE: Results showed that PLD4 is a non-PLD, HKD motif-carrying, transmembrane glycoprotein localized in the endoplasmic reticulum and Golgi apparatus. The spatiotemporally restricted expression patterns suggested that PLD4 might play a role in common function(s) among microglia during early postnatal brain development and splenic marginal zone cells.

Archaeal RNA ligase is a homodimeric protein that catalyzes intramolecular ligation of single-stranded RNA and DNA.

RNA ligases participate in repair, splicing and editing pathways that either reseal broken RNAs or alter their primary structure. Here, we report the characterization of an RNA ligase from the thermophilic archaeon, Methanobacterium thermoautotrophicum. The 381-amino acid Methanobacterium RNA ligase (MthRnl) catalyzes intramolecular ligation of 5'-PO(4) single-strand RNA to form a covalently closed circular RNA molecule through ligase-adenylylate and RNA-adenylylate (AppRNA) intermediates. At the optimal temperature of 65 degrees C, AppRNA was predominantly ligated to a circular product. In contrast, at 35 degrees C, phosphodiester bond formation was suppressed and the majority of the AppRNA was deadenylylated. Sedimentation analysis indicates that MthRnl is a homodimer in solution. The C-terminal 127-amino acid segment is required for dimerization, is itself capable of oligomeization and acts in trans to inhibit the ligation activity of native MthRnl. MthRnl can also join single-stranded DNA to form a circular molecule. The lack of specificity for RNA and DNA by MthRnl may exemplify an undifferentiated ancestral stage in the evolution of ATP-dependent ligases.