Protein kinase CK2 modulates the activity of Maf-family bZIP transcription factor NRL in rod photoreceptors of mammalian retina.

Maf-family basic motif leucine zipper protein NRL specifies rod photoreceptor cell fate during retinal development and, in concert with homeodomain protein CRX and other regulatory factors, controls the expression of most rod-expressed genes including the visual pigment gene Rhodopsin (Rho). Transcriptional regulatory activity of NRL is modulated by post-translational modifications, especially phosphorylation, and mutations at specific phosphosites can lead to retinal degeneration. During our studies to elucidate NRL-mediated transcriptional regulation, we identified protein kinase CK2 in NRL-enriched complexes bound to Rho promoter-enhancer regions and in NRL-enriched high molecular mass fractions from the bovine retina. The presence of CK2 in NRL complexes was confirmed by co-immunoprecipitation from developing and adult mouse retinal extracts. In vitro kinase assay and bioinformatic analysis indicated phosphorylation of NRL at Ser117 residue by CK2. Co-transfection of Csnk2a1 cDNA encoding murine CK2 with human NRL and CRX reduced the bovine Rho promoter-driven luciferase expression in HEK293 cells and mutagenesis of NRL-Ser117 residue to Ala restored the reporter gene activity. In concordance, overexpression of CK2 in the mouse retina in vivo by electroporation resulted in reduction of Rho promoter-driven DsRed reporter expression as well as the transcript level of many phototransduction genes. Thus, our studies demonstrate that CK2 can phosphorylate Ser117 of NRL. Modulation of NRL activity by CK2 suggests intricate interdependence of transcriptional and signaling pathways in maintaining rod homeostasis.

Skin needling with 100% trichloroacetic acid vs. needling with bleomycin in the treatment of recalcitrant cutaneous warts.

The treatment of warts has always been a challenging prospect for dermatologists. In some cases, these warts can become resistant or recalcitrant to treatment. Although a plethora of therapeutic and destructive options is available for wart management, to date no treatment has been found to be completely effective because none of the agents induce specific antiviral immunity. We conducted a study to evaluate the efficacy and safety of skin needling with topical 100% trichloroacetic acid (TCA) against the same type of skin needling with bleomycin in patients with recalcitrant cutaneous warts. In total, 33 (63.5%) patients in the TCA group and 35 (81.4%) in the bleomycin group had complete clearance of all the warts, which was not statistically significant (P = 0.13). There was also no statistically significant difference between the treated and untreated warts in the bleomycin group, whereas in the TCA group there was a significantly higher response rate in the treated warts. The most common adverse event (AE) in both groups was transient procedure site pain. We found that the use of needling plus TCA leads to a faster resolution of warts compared with needling plus bleomycin, with a comparable safety profile. Additionally, we found that TCA is superior to bleomycin for management of multiple warts. However, needling with either TCA or bleomycin has excellent and fairly comparable efficacy, and these methods should be used for the management of multiple or recalcitrant warts, as they have minimal AEs and recurrence rates.

Rlip76: An Unexplored Player in Neurodegeneration and Alzheimer's Disease?

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and is the most common cause of dementia in older people. AD is associated with the loss of synapses, oxidative stress, mitochondrial structural and functional abnormalities, microRNA deregulation, inflammatory responses, neuronal loss, accumulation of amyloid-beta (Aß) and phosphorylated tau (p-tau). AD occurs in two forms: early onset, familial AD and late-onset, sporadic AD. Causal factors are still unknown for a vast majority of AD patients. Genetic polymorphisms are proposed to contribute to late-onset AD via age-dependent increases in oxidative stress and mitochondrial abnormalities. Recent research from our lab revealed that reduced levels of Rlip76 induce oxidative stress, mitochondrial dysfunction and synaptic damage, leading to molecular and behavioral phenotypes resembling late-onset AD. Rlip76 is a multifunctional 76 kDa protein encoded by the RALBP1 gene, located on chromosome 18. Rlip is a stress-protective ATPase of the mercapturic acid pathway that couples clathrin-dependent endocytosis with the efflux of glutathione-electrophile conjugates. Rlip is evolutionarily highly conserved across species and is ubiquitously expressed in all tissues, including AD-affected brain regions, the cerebral cortex and hippocampus, where highly active neuronal metabolisms render the cells highly susceptible to intracellular oxidative damage. In the current article, we summarize molecular and cellular features of Rlip and how depleted Rlip may exacerbate oxidative stress, mitochondrial dysfunction and synaptic damage in AD. We also discuss the possible role of Rlip in aspects of learning and memory via axonal growth, dendritic remodeling, and receptor regulation. We conclude with a discussion of the potential for the contribution of genetic polymorphisms in Rlip to AD progression and the potential for Rlip-based therapies.

Prevalence of Escherichia albertii in Raccoons (Procyon lotor), Japan.

Natural reservoirs of Escherichia albertii remain unclear. In this study, we detected E. albertii by PCR in 248 (57.7%) of 430 raccoons from Osaka, Japan, and isolated 143 E. albertii strains from the 62 PCR-positive samples. These data indicate that raccoons could be a natural reservoir of E. albertii in Japan.

A New Derivatization Reagent for HPLC-MS Analysis of Biological Organic Acids.

Small molecules containing carboxylic acid functional groups are ubiquitous throughout biology, playing vital roles in biological chemistry ranging from energy metabolism to cellular signaling. This paper describes a new derivatization reagent, 4-bromo-N-methylbenzylamine, which was selected for its potential to derivatize mono-, di- and tri-carboxylic acids, such as the intermediates of the tricarboxylic acid (TCA) cycle. This derivatization procedure facilitated the use of positive electrospray ionization (ESI) tandem mass spectrometry (MS/MS) detection of derivatized species allowing for clear identification thanks to the easily recognizable isotope pattern of the incorporated bromine. A liquid chromatography (LC)-MS/MS method was developed which provided limits of detection between 0.2 and 44 µg L-1 in under 6 min, depending on the analyte and total analysis time. This method was successfully applied in both in vitro and in vivo models.

The transcription-splicing protein NonO/p54nrb and three NonO-interacting proteins bind to distal enhancer region and augment rhodopsin expression.

Phototransduction machinery in vertebrate photoreceptors is contained within the membrane discs of outer segments. Daily renewal of 10% of photoreceptor outer segments requires stringent control of gene expression. Rhodopsin constitutes over 90% of the protein in rod discs, and its altered expression or transport is associated with photoreceptor dysfunction and/or death. Two cis-regulatory sequences have been identified upstream of the rhodopsin transcription start site. While the proximal promoter binds to specific transcription factors, including NRL and CRX, the rhodopsin enhancer region (RER) reportedly contributes to precise and high-level expression of rhodopsin in vivo. Here, we report the identification of RER-bound proteins by mass spectrometry. We validate the binding of NonO (p54(nrb)), a protein implicated in coupling transcription to splicing, and three NonO-interacting proteins-hnRNP M, Ywhaz and Ppp1ca. NonO and its interactors can activate rhodopsin promoter in HEK293 cells and function synergistically with NRL and CRX. DNA-binding domain of NonO is critical for rhodopsin promoter activation. Chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) analysis demonstrates high occupancy of NonO at rhodopsin and a subset of phototransduction genes. Furthermore, shRNA knockdown of NonO in mouse retina leads to loss of rhodopsin expression and rod cell death, which can be partially rescued by a C-terminal NonO construct. RNA-seq analysis of the NonO shRNA-treated retina revealed splicing defects and altered expression of genes, specifically those associated with phototransduction. Our studies identify an important contribution of NonO and its interacting modulator proteins in enhancing rod-specific gene expression and controlling rod homeostasis.

Regulation of a novel isoform of Receptor Expression Enhancing Protein REEP6 in rod photoreceptors by bZIP transcription factor NRL.

The Maf-family leucine zipper transcription factor NRL is essential for rod photoreceptor development and functional maintenance in the mammalian retina. Mutations in NRL are associated with human retinopathies, and loss of Nrl in mice leads to a cone-only retina with the complete absence of rods. Among the highly down-regulated genes in the Nrl(-/-) retina, we identified receptor expression enhancing protein 6 (Reep6), which encodes a member of a family of proteins involved in shaping of membrane tubules and transport of G-protein coupled receptors. Here, we demonstrate the expression of a novel Reep6 isoform (termed Reep6.1) in the retina by exon-specific Taqman assay and rapid analysis of complementary deoxyribonucleic acid (cDNA) ends (5'-RACE). The REEP6.1 protein includes 27 additional amino acids encoded by exon 5 and is specifically expressed in rod photoreceptors of developing and mature retina. Chromatin immunoprecipitation assay identified NRL binding within the Reep6 intron 1. Reporter assays in cultured cells and transfections in retinal explants mapped an intronic enhancer sequence that mediated NRL-directed Reep6.1 expression. We also demonstrate that knockdown of Reep6 in mouse and zebrafish resulted in death of retinal cells. Our studies implicate REEP6.1 as a key functional target of NRL-centered transcriptional regulatory network in rod photoreceptors.

Antioxidant role of glutathione S-transferases: 4-Hydroxynonenal, a key molecule in stress-mediated signaling.

4-Hydroxy-2-trans-nonenal (4HNE), one of the major end products of lipid peroxidation (LPO), has been shown to induce apoptosis in a variety of cell lines. It appears to modulate signaling processes in more than one way because it has been suggested to have a role in signaling for differentiation and proliferation. It has been known that glutathione S-transferases (GSTs) can reduce lipid hydroperoxides through their Se-independent glutathione-peroxidase activity and that these enzymes can also detoxify LPO end-products such as 4HNE. Available evidence from earlier studies together with results of recent studies in our laboratories strongly suggests that LPO products, particularly hydroperoxides and 4HNE, are involved in the mechanisms of stress-mediated signaling and that it can be modulated by the alpha-class GSTs through the regulation of the intracellular concentrations of 4HNE. We demonstrate that 4HNE induced apoptosis in various cell lines is accompanied with c-Jun-N-terminal kinase (JNK) and caspase-3 activation. Cells exposed to mild, transient heat or oxidative stress acquire the capacity to exclude intracellular 4HNE at a faster rate by inducing GSTA4-4 which conjugates 4HNE to glutathione (GSH), and RLIP76 which mediates the ATP-dependent transport of the GSH-conjugate of 4HNE (GS-HNE). The balance between formation and exclusion promotes different cellular processes - higher concentrations of 4HNE promote apoptosis; whereas, lower concentrations promote proliferation. In this article, we provide a brief summary of the cellular effects of 4HNE, followed by a review of its GST-catalyzed detoxification, with an emphasis on the structural attributes that play an important role in the interactions with alpha-class GSTA4-4. Taken together, 4HNE is a key signaling molecule and that GSTs being determinants of its intracellular concentrations, can regulate stress-mediated signaling, are reviewed in this article.

Rapid diagnosis and simultaneous identification of tuberculous and bacterial meningitis by a newly developed duplex polymerase chain reaction.

The present study describes the development and evaluation of a duplex polymerase chain reaction (D-PCR) for diagnosis and simultaneous identification of tuberculous meningitis (TBM) and bacterial meningitis (BM) in a single reaction. A D-PCR with primers amplifying portions of the Mycobacterium tuberculosis IS6110 and the eubacteria 16SrDNA sequence in a same reaction mix was developed and tested on DNA extracted from 150 clinical CSF samples from different categories (TBM = 39, BM = 26, control infectious and non-infectious category = 85). The results indicate a clear differentiation between bands for eubacteria and M. tuberculosis with an analytical sensitivity of 10(3) cfu/ml for eubacteria and 10(2) cfu/ml for M. tuberculosis. When evaluated in clinical samples, D-PCR overall diagnosed 100 % confirmed TBM and 100 % confirmed BM cases with overall specificity of 96.5 %. D-PCR can be an effective tool for diagnosis and simultaneous identification of TBM or BM in a single PCR reaction. It saves time, cost, labour and sample amount and help in administration of appropriate antimicrobial therapy. The proposed diagnostic assay would be helpful in correct and rapid management of TBM and BM patients.

The role of RLIP76 in oxidative stress and mitochondrial dysfunction: Evidence based on autopsy brains from Alzheimer's disease patients.

Several converging lines of evidence from our group support a potential role of RLIP76 (AKA Rlip) in neurodegenerative disorders, including Alzheimer's Disease (AD). However, the role of Rlip in Alzheimer's and other neurodegenerative diseases is not well understood. The purpose of the present study is to determine the role of Rlip in the brains of AD patients and control subjects. To achieve our goals, we used frozen tissues and formalin-fixed paraffin-embedded postmortem brains from AD patients of different Braak stages and age-matched control subjects. Our immunohistology and immunoblotting blotting analysis revealed that expression of Rlip protein gradually and significantly decreased (p = 0.0001) with AD progression, being lowest in Braak stage IV-V. Rlip was colocalized with Amyloid beta (Aß) and phosphorylated tau (p-Tau) as observed by IHC staining and co-immunoprecipitation studies. Lipid peroxidation (4-HNE generation) and H2O2 production were significantly higher (p = 0.004 and 0.0001 respectively) in AD patients compared to controls, and this was accompanied by lower ATP production in AD (p = 0.0009). Oxidative DNA damage was measured by 8-Hydroxyguanosine (8-OHdG) in tissue lysates by ELISA and COMET assay. AD 8-OHdG levels were significantly higher (p = 0.0001) compared to controls. COMET assay was performed in brain cells, isolated from frozen postmortem samples. The control samples showed minimal DNA in comets representing few DNA strand breaks (<20 %), (score-0-1). However, the AD group showed an average of 50 % to 65 % of DNA in comet tails (score-4-5) indicating numerous DNA strand breaks. The difference between the two groups was significant (p = 0.001), as analyzed by Open Comet by ImageJ. Elevated DNA damage was further examined by western blot analysis for phosphorylated histone variant H2AX (γH2AX). Induction of γH2AX was very significant (p < 0.0001) and confirmed the presence of double-strand breaks in DNA. Overall, our results indicate an important role for Rlip in maintaining neuronal health and homeostasis by suppressing cellular oxidative stress and DNA damage. Based on our findings, we cautiously conclude that Rlip is a promising therapeutic target for Alzheimer's disease.

Correction: Reddy et al. Rlip Reduction Induces Oxidative Stress and Mitochondrial Dysfunction in Mutant Tau-Expressed Immortalized Hippocampal Neurons: Mechanistic Insights. Cells 2023, 12, 1646.

The authors wish to make the following changes to their paper [...].

Rlip overexpression reduces oxidative stress and mitochondrial dysfunction in Alzheimer's disease: Mechanistic insights.

Alzheimer's disease (AD) is a neurodegenerative disease that affects a large proportion of the aging population. RalBP1 (Rlip) is a stress-activated protein that plays a crucial role in oxidative stress and mitochondrial dysfunction in aging and neurodegenerative diseases but its precise role in the progression of AD is unclear. The purpose of our study is to understand the role of Rlip in the progression and pathogenesis of AD in mutant APP/amyloid beta (Aß)-expressed mouse primary hippocampal (HT22) hippocampal neurons. In the current study, we used HT22 neurons that express mAPP, transfected with Rlip-cDNA and/or RNA silenced, and studied cell survival, mitochondrial respiration, mitochondrial function, immunoblotting & immunofluorescence analysis of synaptic and mitophagy protein's and colocalization of Rlip and mutant APP/Aß proteins and mitochondrial length and number. We also assessed Rlip levels in autopsy brains from AD patients and control subjects. We found cell survival was decreased in mAPP-HT22 cells and RNA-silenced HT22 cells. However, cell survival was increased in Rlip-overexpressed mAPP-HT22 cells. Oxygen consumption rate (OCR) was decreased in mAPP-HT22 cells and RNA-silenced Rlip-HT22 cells. OCR was increased in Rlip-overexpressed in mAPP-HT22 cells. Mitochondrial function was defective in mAPP-HT22 cells and RNA silenced Rlip in HT22 cells, however, it was rescued in Rlip overexpressed mAPP-HT22 cells. Synaptic and mitophagy proteins were decreased in mAPP-HT22 cells, further reducing RNA-silenced Rlip-HT22 cells. However, these were increased in mAPP+Rlip-HT22 cells. Colocalization analysis revealed Rlip is colocalized with mAPP/Aß. An increased number of mitochondria and decreased mitochondrial length were found in mAPP-HT22 cells. These were rescued in Rlip overexpressed mAPP-HT22 cells. Reduced Rlip levels were found in autopsy brains from AD patients. These observations strongly suggest that Rlip deficiency causes oxidative stress/mitochondrial dysfunction and Rlip overexpression reduced these defects.

Rlip Reduction Induces Oxidative Stress and Mitochondrial Dysfunction in Mutant Tau-Expressed Immortalized Hippocampal Neurons: Mechanistic Insights.

RalBP1 (Rlip) is a stress-activated protein that is believed to play a large role in aging and neurodegenerative diseases such as Alzheimer's disease (AD) and other tauopathies. The purpose of our study was to understand the role of Rlip in mutant Tau-expressed immortalized hippocampal HT22 cells. In the current study, we used mutant Tau (mTau)-expressed HT22 neurons and HT22 cells transfected with Rlip-cDNA and/or silenced RNA, and studied the cell survival, mitochondrial respiration, mitochondrial function, immunoblotting, and immunofluorescence analysis of synaptic and mitophagy proteins and the colocalization of Rlip and mTau proteins. We found Rlip protein levels were reduced in mTau-HT22 cells, Rlip silenced HT22 cells, and mTau + Rlip RNA silenced HT22 cells; on the other hand, increased Rlip levels were observed in Rlip cDNA transfected HT22 cells. We found cell survival was decreased in mTau-HT22 cells and RNA-silenced HT22 cells. However, cell survival was increased in Rlip-overexpressed mTau-HT22 cells. A significantly reduced oxygen consumption rate (OCR) was found in mTau-HT22 cells and in RNA-silenced Rlip-HT22 cells, with an even greater reduction in mTau-HT22 + Rlip RNA-silenced HT22 cells. A significantly increased OCR was found in Rlip-overexpressed HT22 cells and in all groups of cells that overexpress Rlip cDNA. Mitochondrial function was defective in mTau-HT22 cells, RNA silenced Rlip in HT22 cells, and was further defective in mTau-HT22 + Rlip RNA-silenced HT22 cells; however, it was rescued in Rlip overexpressed in all groups of HT22 cells. Synaptic and mitophagy proteins were decreased in mTau-HT22 cells, and further reductions were found in RNA-silenced mTau-HT22 cells. However, these were increased in mTau + Rlip-overexpressed HT22 cells. An increased number of mitochondria and decreased mitochondrial length were found in mTau-HT22 cells. These were rescued in Rlip-overexpressed mTau-HT22 cells. These observations strongly suggest that Rlip deficiency causes oxidative stress/mitochondrial dysfunction and Rlip overexpression reverses these defects. Overall, our findings revealed that Rlip is a promising new target for aging, AD, and other tauopathies/neurological diseases.

Leveraging cryoablation and checkpoint inhibitors for high-risk triple negative breast cancer.

Breast cancer is the second most common cancer among women in the United States in which the standard of care treatment is surgery with adjunctive therapy. Cryoablation, which destroys the tumor using extremely cold temperatures while preserving the potential tumor antigens, is a promising alternative to surgical resection. It is less invasive, cosmetically appeasing, cost-effective, and capable of contributing to the abscopal effect - the immune response targeting potential distant metastasis. However, to maximize the immunologic benefit of cryoablation in biologically high-risk breast cancers, combination with therapies that enhance immune activation, such as immune checkpoint inhibitors (ICIs) may be necessary. This mini review describes the fundamentals of cryoablation and treatment with ICIs, as well as discuss the caveats in both strategies and current clinical trials aimed to improve this approach to benefit patients.

Effects of proton and oxygen ion irradiation on cardiovascular function and structure in a rabbit model.

PURPOSE: Astronauts on missions beyond low Earth orbit will be exposed to galactic cosmic radiation, and there is concern about potential adverse cardiovascular effects. Most of the research to identify cardiovascular risk of space radiation has been performed in rodent models. To aid in the translation of research results to humans, the current study identified long-term effects of high-energy charged particle irradiation on cardiovascular function and structure in a larger non-rodent animal model. MATERIALS AND METHODS: At the age of 12 months, male New Zealand white rabbits were exposed to whole-body protons (250 MeV) or oxygen ions (16O, 600 MeV/n) at a dose of 0 or 0.5 Gy and were followed for 12 months after irradiation. Ultrasonography was used to measure in vivo cardiac function and blood flow parameters at 10- and 12-months post-irradiation. At 12 months after irradiation, blood cell counts and blood chemistry values were assessed, and cardiac tissue and aorta were collected for histological as well as molecular and biochemical analyses. Plasma was used for metabolomic analysis and to quantify common markers of cardiac injury. RESULTS: A small but significant decrease in the percentage of circulating lymphocytes and an increase in neutrophil percentage was seen 12 months after 0.5 Gy protons, while 16O exposure resulted in an increase in monocyte percentage. Markers of cardiac injury, cardiac troponin I (cTnI) and N-Terminal pro-B-type Natriuretic Peptide were modestly increased in the proton group, and cTnI was also increased after 16O. On the other hand, metabolomics on plasma at 12 months revealed no changes. Both types of irradiation demonstrated alterations in cardiac mitochondrial morphology and an increase in left ventricular protein levels of inflammatory cell marker CD68. However, changes in cardiac function were only mild. CONCLUSION: Low dose charged particle irradiation caused mild long-term changes in inflammatory markers, cardiac function, and structure in the rabbit heart, in line with previous studies in mouse and rat models.

Rlip Protein: A Potential Target for COVID-19.

On January 30, 2020, the COVID-19 epidemic was declared an international public health emergency by the World Health Organization. Given the growing impact of the pandemic, there is great interest in finding potential targets for treating infected or hospitalized COVID-19 patients. Therapeutic studies have been conducted on pre-existing drugs, which vary by country, including anti-malarial agents, antiviral agents, and convalescent plasma. However, many of these agents are ineffective at reducing mortality or only shorten the severity or duration of COVID-19 illness in hospitalized patients. As such, other alternatives for treating COVID-19 are being investigated. One such target of interest has been clathrin-dependent endocytosis (CDE). Clathrin-dependent endocytosis is the most commonly observed mechanism of viral entry into cells. However, there have been no published studies to date on CDE inhibition strategies against COVID-19. One such target is Rlip or RLIP76 (human gene RALBP1, 18p11.22). Among its many functions, Rlip is a stress-protective, Ral-regulated ATPase of the mercapturic acid pathway that transports glutathione-electrophile conjugates of electrophilic toxins, which are precursors of mercapturic acid that precedes de-glutamylation by gamma-glutamyl transferase. Rlip is also regulated by several G-proteins that coordinate movement of cells, organelles, membranes, cytoskeleton, macromolecules, and other small molecules. Previous studies have link Rlip in the pathogenesis of several viral illness. In this paper, we want to propose that RLIP76 (Rlip or RALBP1) may be a novel target for treating SARS-CoV-2 viral infections.

Rlip Depletion Alters Oncogene Transcription at Multiple Distinct Regulatory Levels.

Rlip76 (Rlip) is a multifunctional membrane protein that facilitates the high metabolic rates of cancer cells through the efflux of toxic metabolites and other functions. Rlip inhibition or depletion results in broad-spectrum anti-cancer effects in vitro and in vivo. Rlip depletion effectively suppresses malignancy and causes global reversion of characteristic CpG island methylomic and transcriptomic aberrations in the p53-null mouse model of spontaneous carcinogenesis through incompletely defined signaling and transcriptomic mechanisms. The methylome and transcriptome are normally regulated by the concerted actions of several mechanisms that include chromatin remodeling, promoter methylation, transcription factor interactions, and miRNAs. The present studies investigated the interaction of Rlip depletion or inhibition with the promoter methylation and transcription of selected cancer-related genes identified as being affected by Rlip depletion in our previous studies. We constructed novel promoter CpG island/luciferase reporter plasmids that respond only to CpG methylation and transcription factors. We found that Rlip depletion regulated expression by a transcription factor-based mechanism that functioned independently of promoter CpG methylation, lipid peroxidation, and p53 status.

The human hGSTA5 gene encodes an enzymatically active protein.

BACKGROUND: Of the five human Alpha-class glutathione transferases, expression of hGSTA5 has not been experimentally documented, even though in silico the hGSTA5 sequence can be assembled into a mRNA and translated. The present work was undertaken to determine whether hGSTA5 is functional. METHODS: Human K562 cells were transfected with the hGSTA5 gene driven by the CMV promoter, and hGSTA5 cDNA was recovered from mature mRNA by reverse transcription. The cDNA was used in bacterial and eukaryotic protein expression systems. The resulting protein, after purification by glutathione affinity chromatography where appropriate, was tested for glutathione transferase activity. RESULTS: Human K562 cells transfected with the hGSTA5 gene under control of a CMV promoter produced a fully spliced mRNA which, after reverse transcription and expression in E. coli, yielded a protein that catalyzed the conjugation of the lipid peroxidation product 4-hydroxynonenal to glutathione. Similarly, transfection of human HEK-293 cells with the hGSTA5 gene driven by the CMV promoter led to an elevated 4-hydroxynonenal-conjugating activity in the cell lysate. In addition, translation of hGSTA5 cDNA in a cell-free eukaryotic system gave rise to a protein with 4-hydroxynonenal-conjugating activity. CONCLUSIONS: hGSTA5 can be processed to a mature mRNA which is translation-competent, producing a catalytically active enzyme. GENERAL SIGNIFICANCE: Because a functional gene would not be maintained in the absence of selective pressure, we conclude that the native hGSTA5 promoter is active but has a spatially or temporally restricted expression pattern, and/or is expressed only under specific (patho)physiological conditions.

Moraxella catarrhalis: an unrecognized pathogen of the oral cavity?

OBJECTIVE: We investigated the effect of the bacterial flora of the nose and throat on the outcome of the initial repairs of the cleft palate in the presence of prophylactic antibiotics. DESIGN: A retrospective review of 90 procedures in 66 patients who had cleft palate repair between April 2005 and June 2007 was conducted at Booth Hall Children's Hospital, Manchester, U.K. Both isolated cleft palate and cleft lip and palate patients were included. Exclusion criteria included syndromic cases, other medical disorders, and revisions of previous cleft palate repairs. Nose and throat swabs were taken on admission. Benzyl penicillin and flucloxacillin were given perioperatively. The occurrence of oronasal fistulas was correlated with the bacteria grown on culture. RESULTS: The oronasal fistula rate was 15.9%. The highest fistula rate in procedures with positive swabs was seen with Moraxella catarrhalis. CONCLUSIONS: M. catarrhalis has not been previously recognized as a pathogen in cleft palate repairs. This study demonstrates a higher fistula rate in procedures positive for M. catarrhalis. Other factors that may have contributed to the fistula formation include the severity of the initial cleft and technical factors. Further study is required before a definitive link can be established.