Deficient Variants Research Articles

Abstract 1742: Modulation of genotoxic DNA damage by the ATPase domain of type II topoisomerases

Abstract Type II topoisomerases play key roles in transcription, replication and chromosome segregation. These enzymes modulate DNA structure by generating transient DNA breaks where the protein is covalently bound to DNA. Anti-cancer drugs targeting topoisomerase II (Top2) inhibit the religation of enzyme-induced breaks, and exert their anti-cancer effects through the generation of enzyme mediated DNA damage. In addition to small molecule inhibitors, it has been suggested that aberrant DNA structures or DNA lesions may also lead to Top2/DNA lesions. A mammalian Top2 isoform, Top2β has been suggested to play a key role in hormone-mediated transcription, and in carrying out this function, generates potentially oncogenic DNA damage. We have assessed the generation of potentially oncogenic DNA damage by Top2β in model organisms, and using the purified protein. We found that ectopic expression of Top2β is poorly tolerated in repair deficient yeast strains, while expression of the mammalian Top2α isoform lacks this property. We measured the induction of homologous recombination by Top2β in yeast and found that ectopic expression of the enzyme led to elevated levels of recombination. Recently, a de novo mutation in Top2β was reported in a patient with severe developmental delay resulting from an His63Tyr substitution in Top2β 1. We found that Top2β (His63Tyr) could not complement a defect in yeast Top2, but encoded an enzyme that was catalytically active, and induced elevated levels of recombination. Because of the inferred location of His63 in the ATPase domain of Top2β, and proximity to the ATP binding site of the enzyme, we hypothesized that the mutant protein was partially defective in ATP regulated DNA cleavage. To further explore how ATP regulates Top2β cleavage, we examined the effects of expressing a Top2β variant completely lacking the N-terminal ATPase domain of Top2β. While this mutant did not complement yeast top2 mutants, we found that it s expression induced 10-20 fold higher levels of recombination than cells expressing wild type Top2β. This result indicates that an ATP deficient variant of Top2β induces very high levels of DNA damage. We suggest that regulation of the ATPase activity of Top2β may be critical for maintaining genome stability, and may be responsible for induction of DNA damage during hormone-mediated transcription. 1. C.W. Lam et al., Global developmental delay and intellectual disability associated with a de novo TOP2B mutation. Clinica Chimica Acta 469:63-68, 2017. Citation Format: Amanda M. Johnson, Lokha Ranjani A. Boopathy, Raveena Gupta, Hannah N. Miles, Matthew Gilbertson, Karin C. Nitiss, John L. Nitiss. Modulation of genotoxic DNA damage by the ATPase domain of type II topoisomerases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1742.

Safety and tolerability of single low-dose primaquine in a low-intensity transmission area in South Africa: an open-label, randomized controlled trial

BackgroundTo reduce onward falciparum malaria transmission, the World Health Organization recommends adding single low-dose (SLD) primaquine to artemisinin-based combination treatment in low transmission areas. However, uptake of this recommendation has been relatively slow given concerns about whether individual risks justify potential community benefit. This study was undertaken to generate comprehensive local data on the risk–benefit profile of SLD primaquine deployment in a pre-elimination area in South Africa.MethodsThis randomized, controlled open-label trial investigated adding a single low primaquine dose on day 3 to standard artemether–lumefantrine treatment for uncomplicated falciparum malaria. Efficacy, safety and tolerability of artemether–lumefantrine and primaquine treatment were assessed on days 3, 7, 14, 28 and 42. Lumefantrine concentrations were assayed from dried blood spot samples collected on day 7.ResultsOf 217 patients screened, 166 were enrolled with 140 randomized on day 3, 70 to each study arm (primaquine and no primaquine). No gametocytes were detected by either microscopy or PCR in any of the follow-up samples collected after randomization on day 3, precluding assessment of primaquine efficacy. Prevalence of the CYP2D6*4, CYP2D6*10 and CYP2D6*17 mutant alleles was low with allelic frequencies of 0.02, 0.11 and 0.16, respectively; none had the CYP2D6*4/*4 variant associated with null activity. Among 172 RDT-positive patients G6PD-genotyped, 24 (14%) carried the G6PD deficient (A−) variant. Median haemoglobin concentrations were similar between treatment arms throughout follow-up. A third of participants had a haemoglobin drop > 2 g/dL; this was not associated with primaquine treatment but may be associated with G6PD genotype [52.9% (9/17) with A− genotype vs. 31% (36/116) with other genotypes (p = 0.075)]. Day 7 lumefantrine concentrations and the number and nature of adverse events were similar between study arms; only one serious adverse event occurred (renal impairment in the no primaquine arm). The artemether–lumefantrine PCR-corrected adequate clinical and parasitological response rate was 100%, with only one re-infection found among the 128 patients who completed 42-day follow-up.ConclusionsSafety, tolerability, CYP2D6 and G6PD variant data from this study support the deployment of the WHO-recommended SLD primaquine without G6PD testing to advance malaria elimination in South African districts with low-intensity residual transmission.Trial registration Pan African Clinical Trial Registry, PACTR201611001859416. Registered 11 November 2016, https://pactr.samrc.ac.za/TrialDisplay.aspx?TrialID=1859

Identifying membrane-bound quinoprotein glucose dehydrogenase from acetic acid bacteria that produce lactobionic and cellobionic acids.

Acetic acid bacteria are used in the commercial production of lactobionic acid (LacA). However, the lactose-oxidizing enzyme of these bacteria remains unidentified. Lactose-oxidizing activity has been detected in bacterial membrane fractions and is strongly inhibited by d-glucose, suggesting that the enzyme was a membrane-bound quinoprotein glucose dehydrogenase, but these dehydrogenases have been reported to be incapable of oxidizing lactose. Thus, we generated m-GDH-overexpressing and -deficient strains of Komagataeibacter medellinensis NBRC3288 and investigated their lactose-oxidizing activities. Whereas the overexpressing variants produced ~2-5-fold higher amounts of LacA than the wild-type strains, the deficient variant produced no LacA or d-gluconic acid. Our results indicate that the lactose-oxidizing enzyme from acetic acid bacteria is membrane-bound quinoprotein glucose dehydrogenase. Abbreviations: LacA: lactobionic acid; AAB: acetic acid bacterium; m-GDH: membrane-bound quinoprotein glucose dehydrogenase; DCIP: 2,6-dichlorophenolindophenol; HPAEC-PAD: high-performance anion-exchange chromatography with pulsed amperometric detection.

Overview of Alpha-1 Antitrypsin Deficiency-Mediated Liver Disease

Alpha-1 antitrypsin (AAT), encoded by the SERPINA1 gene, is a protein mainly produced and secreted by hepatocytes. Some specific mutations affecting SERPINA1 may cause accumulation of misfolded AAT in the endoplasmic reticulum of the hepatocytes leading to AAT deficiency (AATD). Z-AAT is the most severe and common deficient variant. This mutant is not only retained in the endoplasmic reticulum but accumulates as an aggregate that triggers a cascade of intracellular signalling pathways inducing hepatocyte injury and death. Nevertheless, among all the homozygous ZZ patients only 15% develop liver injury, with a wide-range of disease severities ranging from hepatic fibrosis to cirrhosis or even hepatocellular carcinoma. Due to the lack of knowledge surrounding modifiers associated with Z-AAT-mediated hepatocyte toxicity, it is impossible to screen for AATD patients at risk of liver damage and to develop accurate therapeutic strategies. This review aims to give an overview and update our knowledge of AATD associated with liver disease and discusses possible new therapeutic strategies.

Inherited glycophosphatidylinositol deficiency variant database and analysis of pathogenic variants.

BackgroundGlycophosphatidylinositol‐anchored proteins (GPI‐APs) mediate several physiological processes such as embryogenesis and neurogenesis. Germline variants in genes involved in their synthesis can disrupt normal development and result in a variety of clinical phenotypes. With the advent of new sequencing technologies, more cases are identified, leading to a rapidly growing number of reported genetic variants. With this number expected to rise with increased accessibility to molecular tests, an accurate and up‐to‐date database is needed to keep track of the information and help interpret results.MethodsWe therefore developed an online resource (www.gpibiosynthesis.org) which compiles all published pathogenic variants in GPI biosynthesis genes which are deposited in the LOVD database. It contains 276 individuals and 192 unique public variants; 92% of which are predicted as damaging by bioinformatics tools.ResultsA significant proportion of recorded variants was substitution variants (81%) and resulted mainly in missense and frameshift alterations. Interestingly, five patients (2%) had deleterious mutations in untranslated regions. CADD score analysis placed 97% of variants in the top 1% of deleterious variants in the human genome. In genome aggregation database, the gene with the highest frequency of reported pathogenic variants is PIGL, with a carrier rate of 1/937.ConclusionWe thus present the GPI biosynthesis database and review the molecular genetics of published variants in GPI‐anchor biosynthesis genes.

A positive correlation between sickle cell anemia and g6pd deficiency from population of Chhattisgarh, India

ObjectivePresent study was undertaken to study the association between sickle cell anemia (SCA) and glucose-6-phosphate dehydrogenase (G6PD) deficiency from Sahu and Kurmi population of Durg and Rajnandgaon district of Chhattisgarh, India. MethodA random sampling of 1749 individuals was done. SCA and G6PD deficiency was detected by slide test followed by electrophoresis and Enzymatic reaction indicated by change in colour respectively. Further the samples were subjected to analyze glutathione-S-transferase (GST) i.e. GSTM1 and GSTT1 gene polymorphism, variance of G6PD among G6PD deficient samples by PCR-RFLP. Oxidative stress and DNA damage by comet assay was also analyzed. ResultsPresent finding indicates positive correlation between SCA and G6PD deficiency in Durg and Rajnandgaon district [Durg: (r = 0.92; HbAS-G6PDd and r = 0.56; HbSS-G6PDd) Rajnandgaon: (r = 0.63; HbAS-G6PDd and r = 0.86; HbSS-G6PDd)]. Significant changes (P < 0.05) in antioxidant enzymatic parameters were observed in HbSS and G6PD with sickle positive individual. Assessment of DNA damage by Comet assay considering Head DNA percent, Tail DNA percent, Tail length and Tail moment also showed significant changes (P < 0.05) within all concerned parameters in HbSS and G6PD with sickle positive individual. Analysis of GST gene polymorphism showed that frequency of individuals carrying the GSTM1 null genotype was higher in HbAS (60%) and the frequency of individual carrying the GSTT1 null genotype was found higher in HbSS (66.6%). G6PD variants analysis also confirmed the presence of highest percentage of mutation among G6PD deficient population as compared to control and a positive correlation was observed between G6PD deficiency and mutant variants of G6PD gene [Rajnandgaon: (r = 0.67; G6PDd-Mahidol mutated and r = 0.90; G6PDd-Union mutated) Durg: (r = 0.91; G6PDd-Mahidol mutated and r = 0.01; G6PDd-Union mutated)] . ConclusionThus present finding indicates positive correlation between SCA and G6PD deficiency in Chhattisgarh, India.

Ficolin-3 Deficiency Is Associated with Disease and an Increased Risk of Systemic Lupus Erythematosus.

Ficolin-3 deficiency is caused by a mutation (+1637delC) in the FCN3 gene. It is a rare condition and has been associated with both infection and autoimmune disease including systemic lupus erythematosus (SLE). Here we investigated if ficolin-3 deficiency is more frequent in patients than in controls and tried to identify a common phenotype among ficolin-3 deficient individuals. Since a significant part of patients identified with ficolin-3 deficiency was diagnosed with SLE, we explored whether the heterozygous state of the FCN3+1637delC variant represents a risk factor in the development of SLE. Further, we examined other possible causes of ficolin-3 deficiency when the FCN3+1637delC is not present. A systematic literature search for studies measuring ficolin-3 was carried out. We examined 362 SLE patients and 596 controls for the presence of the variant FCN3+1637delC. We established assays for measurements of ficolin-3 and of auto-antibodies against ficolin-3. We sequenced the coding and non-coding regions of the FCN3 gene in an SLE patient with ficolin-3 deficiency not carrying the +1637delC. Ficolin-3 deficiency leads to an 8-time increased odds of having a disease (p< 0.05). Three out of nine patients with deficiency had SLE. The heterozygous state of the deficiency variant is not associated with increased risk of developing SLE (p= 0.18). By systematically reviewing the literature for the described cases of ficolin-3 deficiency, an autoimmune phenotype is emerging. Thirty-three percent of the ficolin-3 deficient patients had SLE. Heterozygosity for the FCN3 gene deletion causing the deficiency does not seem to be associated with the development of SLE.

088 Skin delivery of modified vaccinia ankara (MVA) virus generates superior protective pulmonary immunity

No viral infection has shaped human history more profoundly than smallpox (Variola major). Although it was eradicated in vivo by a world-wide vaccination program by 1980, it remains a potential threat via bioterrorism, as most of the world’s population has never been immunized. There is an unacceptable incidence of morbidity after vaccinia virus (VACV) vaccination, particularly in individuals with atopic disorders. Therefore, a less virulent replication deficient variant of VACV, called MVA, has been developed. In all prior studies in humans, MVA has been delivered via intramuscular (im) injection. Variola major is transmitted by respiratory droplets, and successful immune response to V. major are T cell rather than antibody mediated. We showed that MVA as low as 1.8 X 106 pfu delivered by skin scarification (ss) elicited strong T cell mediated responses that completely protected mice against lethal respiratory challenge. MVA could be safely inoculated to skin of immunocompromised mice without any morbidity. Nearly equivalent numbers of lung tissue TRM could be generated via MVA ss compared to direct pulmonary infection; both were sufficient to protect mice against lethal pulmonary challenge with WT VACV. Strikingly, MVA ss-generated activated T cells exhibiting similar global gene transcriptional profile with those generated via pulmonary infection. Moreover, comparisons of ss with other vaccine delivery routes employed in clinic (id, sc or im) showed that MVA ss-generated T cells were both more abundant and qualitatively superior. Specifically, MVA ss produced more lung CD8+ TRM and was superior in protecting mice against lethal respiratory challenge. Mice genetically deficient in LC and Langerin+ DC demonstrated that both are required for generating an optimal CD8+ T cell response. Our data show that ss with MVA generates robust pulmonary protective immunity, and could be used as a safe and superior vaccine strategy against smallpox. This may be the preferred mode of delivery for MVA as a vaccine vector for other infectious and malignant diseases.

Human pantothenate kinase 4 is a pseudo-pantothenate kinase.

Pantothenate kinase generates 4'-phosphopantothenate in the first and rate-determining step of coenzyme A (CoA) biosynthesis. The human genome encodes three well-characterized and nearly identical pantothenate kinases (PANK1-3) plus a putative bifunctional protein (PANK4) with a predicted amino-terminal pantothenate kinase domain fused to a carboxy-terminal phosphatase domain. Structural and phylogenetic analyses show that all active, characterized PANKs contain the key catalytic residues Glu138 and Arg207 (HsPANK3 numbering). However, all amniote PANK4s, including human PANK4, encode Glu138Val and Arg207Trp substitutions which are predicted to inactivate kinase activity. Biochemical analysis corroborates bioinformatic predictions-human PANK4 lacks pantothenate kinase activity. Introducing Glu138Val and Arg207Trp substitutions to the human PANK3 and plant PANK4 abolished their robust pantothenate kinase activity. Introducing both catalytic residues back into human PANK4 restored kinase activity, but only to a low level. This result suggests that epistatic changes to the rest of the protein already reduced the kinase activity prior to mutation of the catalytic residues in the course of evolution. The PANK4 from frog, an anamniote living relative encoding the catalytically active residues, had only a low level of kinase activity, supporting the view that HsPANK4 had reduced kinase activity prior to the catalytic residue substitutions in amniotes. Together, our data show that human PANK4 is a pseudo-pantothenate kinase-a catalytically deficient variant of the catalytically active PANK4 found in plants and fungi. The Glu138Val and Arg207Trp substitutions in amniotes (HsPANK3 numbering) completely deactivated the pantothenate kinase activity that had already been reduced by prior epistatic mutations.

Werner syndrome helicase is a selective vulnerability of microsatellite instability-high tumor cells.

Targeted cancer therapy is based on exploiting selective dependencies of tumor cells. By leveraging recent functional screening data of cancer cell lines we identify Werner syndrome helicase (WRN) as a novel specific vulnerability of microsatellite instability-high (MSI-H) cancer cells. MSI, caused by defective mismatch repair (MMR), occurs frequently in colorectal, endometrial and gastric cancers. We demonstrate that WRN inactivation selectively impairs the viability of MSI-H but not microsatellite stable (MSS) colorectal and endometrial cancer cell lines. In MSI-H cells, WRN loss results in severe genome integrity defects. ATP-binding deficient variants of WRN fail to rescue the viability phenotype of WRN-depleted MSI-H cancer cells. Reconstitution and depletion studies indicate that WRN dependence is not attributable to acute loss of MMR gene function but might arise during sustained MMR-deficiency. Our study suggests that pharmacological inhibition of WRN helicase function represents an opportunity to develop a novel targeted therapy for MSI-H cancers.

Prevalence of G6PD Viangchan variant in malaria endemic areas in Lao PDR: an implication for malaria elimination by 2030

BackgroundPrimaquine is effective against the latent liver stage of Plasmodium vivax. Eliminating the latent liver stage of P. vivax is one of the necessary conditions to achieve the goal of malaria elimination in Lao People’s Democratic Republic (PDR) by 2030. However, people with glucose-6-phosphate dehydrogenase (G6PD) deficiency are at risk of haemolysis when ingesting primaquine. The aim of this study was to detect the prevalence of the G6PD Viangchan variant, which is said to be common in Lao PDR and which can result in severe haemolysis in patients exposed to primaquine.MethodsBlood samples were collected from villagers in three malaria endemic provinces: Champasak and Savannakhet in the south, and Phongsaly in the north. Each blood sample was semi-quantitatively assayed for G6PD enzyme activity using the G6PD Assay Kit-WST Lyophilized (DOJINDO Laboratories, Japan). Blood samples that were found to be G6PD deficient were sequenced to detect G6PD Viangchan mutation.ResultsIn total, 2043 blood samples were collected from Phongsaly (n = 426, 20.9%), Savannakhet (n = 924, 45.2%), and Champasak (n = 693, 33.9%) provinces in Lao PDR from 2016 to 2017. Of these, 964 (47.2%) were taken from male villagers and 1079 (52.8%) were taken from female villagers. G6PD Viangchan mutation was not detected in Phongsaly province in this study. In Savannakhet province, 48 of the 924 samples (45 males, 3 females) had the G6PD Viangchan mutation (n = 48, 5.2%). In Champasak province, 42 of the 693 samples (18 males, 24 females) had the G6PD Viangchan mutation (n = 42, 6.1%).ConclusionsG6PD Viangchan variant, which can cause severe haemolysis in the carrier when exposed to primaquine, was detected among 6.1% of the villagers in Champasak and 5.2% in Savannakhet but not in Phongsaly in this study. G6PD Viangchan variant might be common in the south of Laos but not so in the north. In the north, other G6PD deficiency variants might be more prevalent. However, in order not to overlook anyone and ensure a safe primaquine therapy for people living in malaria endemic areas in Lao PDR, G6PD testing is necessary.

Cell-Free Expression and Photo-Crosslinking of the Human Neuropeptide Y2 Receptor.

G protein-coupled receptors (GPCRs) represent a large family of different proteins, which are involved in physiological processes throughout the entire body. Furthermore, they represent important drug targets. For rational drug design, it is important to get further insights into the binding mode of endogenous ligands as well as of therapeutic agents at the respective target receptors. However, structural investigations usually require homogenous, solubilized and functional receptors, which is still challenging. Cell-free expression methods have emerged in the last years and many different proteins are successfully expressed, including hydrophobic membrane proteins like GPCRs. In this work, an Escherichia coli based cell-free expression system was used to express the neuropeptide Y2 receptor (Y2R) for structural investigations. This GPCR was expressed in two different variants, a C-terminal enhanced green fluorescent fusion protein and a cysteine deficient variant. In order to obtain soluble receptors, the expression was performed in the presence of mild detergents, either Brij-35 or Brij-58, which led to high amounts of soluble receptor. Furthermore, the influence of temperature, pH value and additives on protein expression and solubilization was tested. For functional and structural investigations, the receptors were expressed at 37°C, pH 7.4 in the presence of 1 mM oxidized and 5 mM reduced glutathione. The expressed receptors were purified by ligand affinity chromatography and functionality of Y2R_cysteine_deficient was verified by a homogenous binding assay. Finally, photo-crosslinking studies were performed between cell-free expressed Y2R_cysteine_deficient and a neuropeptide Y (NPY) analog bearing the photoactive, unnatural amino acid p-benzoyl-phenylalanine at position 27 and biotin at position 22 for purification. After enzymatic digestion, fragments of crosslinked receptor were identified by mass spectrometry. Our findings demonstrate that, in contrast to Y1R, NPY position 27 remains flexible when bound to Y2R. These results are in agreement with the suggested binding mode of NPY at Y2R.

DPYD*6 plays an important role in fluoropyrimidine toxicity in addition to DPYD*2A and c.2846A>T: a comprehensive analysis in 1254 patients

Dihydropyrimidine dehydrogenase (DPYD) is a highly polymorphic gene and classic deficient variants (i.e., c.1236G>A/HapB3, c.1679T>G, c.1905+1G>A and c.2846A>T) are characterized by impaired enzyme activity and risk of severe adverse drug reactions (ADRs) in patients treated with fluoropyrimidines. The identification of poor metabolizers by pre-emptive DPYD screening may reduce the rate of ADRs but many patients with wild-type genotype for classic variants may still display ADRs. Therefore, the search for additional DPYD polymorphisms associated with ADRs may improve the safety of treatment with fluoropyrimidines. This study included 1254 patients treated with fluoropyrimidine-containing regimens and divided into cohort 1, which included 982 subjects suffering from gastrointestinal G≥2 and/or hematological G≥3 ADRs, and cohort 2 (control group), which comprised 272 subjects not requiring dose reduction, delay or discontinuation of treatment. Both groups were screened for DPYD variants c.496A>G, c.1236G>A/HapB3, c.1601G>A (DPYD*4), c.1627A>G (DPYD*5), c.1679T>G (DPYD*13), c.1896T>C, c.1905 + 1G>A (DPYD*2A), c.2194G>A (DPYD*6), and c.2846A>T to assess their association with toxicity. Genetic analysis in the two cohorts were done by Real-Time PCR of DNA extracted from 3 ml of whole blood. DPYD c.496A>G, c.1601G>A, c.1627A>G, c.1896T>C, and c.2194G>A variants were found in both cohort 1 and 2, while c.1905+1G>A and c.2846A>T were present only in cohort 1. DPYD c.1679T>G and c.1236G>A/HapB3 were not found. Univariate analysis allowed the selection of c.1905+1G>A, c.2194G>A and c.2846A>T alleles as significantly associated with gastrointestinal and hematological ADRs (p < 0.05), while the c.496A>G variant showed a positive trend of association with neutropenia (p = 0.06). In conclusion, c.2194G>A is associated with clinically-relevant ADRs in addition to the already known c.1905+1G>A and c.2846A>T variants and should be evaluated pre-emptively to reduce the risk of fluoropyrimidine-associated ADRs.

Transient versus Permanent MCA Occlusion in Mice Genetically Modified to Have Good versus Poor Collaterals.

Collateral-dependent blood flow is capable of significantly lessening the severity of stroke. Unfortunately, collateral flow varies widely in patients for reasons that remain unclear. Studies in mice have shown that the number and diameter of cerebral collaterals vary widely due primarily to polymorphisms in genes, e.g., Rabep2, involved in their formation during development. However, understanding how variation in collateral abundance affects stroke progression has been hampered by lack of a method to reversibly ligate the distal middle cerebral artery (MCAO) in mice. Here we present a method and examine infarct volume 24 h after transient (tMCAO, 90 min) versus permanent occlusion (pMCAO) in mice with good versus poor collaterals. Wildtype C57BL/6 mice (have abundant collaterals) sustained small infarctions following tMCAO that increased 2.1-fold after pMCAO, reflecting significant penumbra present at 90 min. Mutant C57BL/6 mice lacking Rabep2 (have reduced collaterals) sustained a 4-fold increase in infarct volume over WT following tMCAO and a smaller additional increase (0.4-fold) after pMCAO, reflecting reduced penumbra. Wildtype BALB/cBy (have a deficient Rabep2 variant and poor collaterals) had large infarctions following tMCAO that increased less (0.6-fold) than the above wildtype C57BL/6 mice following pMCAO. Mutant BALB/cBy mice (have deficient Rabep2 replaced with the C57BL/6 variant thus increased collaterals) sustained smaller infarctions after tMCAO. However, unlike C57BL/6 versus Rabep2 mice, penumbra was not increased since infarct volume increased only 0.3-fold following pMCAO. These findings present a murine model of tMCAO and demonstrate that neuroprotective mechanisms, in addition to collaterals, also vary with genetic background and affect the evolution of stroke.

ALDH2 and Cardiovascular Disease.

Aldehyde dehydrogenase 2 (ALDH2) is a non-cytochrome P450 mitochondrial aldehyde oxidizing enzyme. It is best known for its role in the metabolism of acetaldehyde, a common metabolite from alcohol drinking. More evidences have been accumulated in recent years to indicate a greater role of ALDH2 in the metabolism of other endogenous and exogenous aldehydes, especially lipid peroxidation-derived reactive aldehyde under oxidative stress. Many cardiovascular diseases are associated with oxidative stress and mitochondria dysfunction. Considering that an estimated 560million East Asians carry a common ALDH2 deficient variant which causes the well-known alcohol flushing syndrome due to acetaldehyde accumulation, the importance of understanding the role of ALDH2 in these diseases should be highlighted. There are several unfavorable cardiovascular conditions that are associated with ALDH2 deficiency. This chapter reviews the function of ALDH2 in various pathological conditions of the heart in relation to aldehyde toxicity. It also highlights the importance and clinical implications of interaction between ALDH2 deficiency and alcohol drinking on cardiovascular disease among the East Asians.

The Effect of Glucose 6-Phosphate Dehydrogenase Deficiency (G6PDd) and Haemoglobin Variants on Malaria in Port Harcourt, Rivers State, Nigeria

The Effect of Glucose 6-Phosphate Dehydrogenase Deficiency (G6PDd) and Haemoglobin Variants on Malaria in Port Harcourt, Rivers State, Nigeria

HIV-1 Nef Disrupts CD4+ T Lymphocyte Polarity, Extravasation, and Homing to Lymph Nodes via Its Nef-Associated Kinase Complex Interface.

HIV-1 Nef is a multifunctional protein that optimizes virus spread and promotes immune evasion of infected cells to accelerate disease progression in AIDS patients. As one of its activities, Nef reduces the motility of infected CD4+ T lymphocytes in confined space. In vivo, Nef restricts T lymphocyte homing to lymph nodes as it reduces the ability for extravasation at the diapedesis step. Effects of Nef on T lymphocyte motility are typically mediated by its ability to reduce actin remodeling. However, interference with diapedesis does not depend on residues in Nef required for inhibition of host cell actin dynamics. In search for an alternative mechanism by which Nef could alter T lymphocyte extravasation, we noted that the viral protein interferes with the polarization of primary human CD4+ T lymphocytes upon infection with HIV-1. Expression of Nef alone is sufficient to disrupt T cell polarization, and this effect is conserved among lentiviral Nef proteins. Nef acts by arresting the oscillation of CD4+ T cells between polarized and nonpolarized morphologies. Mapping studies identified the binding site for the Nef-associated kinase complex (NAKC) as critical determinant of this Nef activity and a NAKC-binding-deficient Nef variant fails to impair CD4+ T lymphocyte extravasation and homing to lymph nodes. These results thus imply the disruption of T lymphocyte polarity via its NAKC binding site as a novel mechanism by which lentiviral Nef proteins alter T lymphocyte migration in vivo.

Noncanonical role for the binding protein in substrate uptake by the MetNI methionine ATP Binding Cassette (ABC) transporter

The Escherichia coli methionine ABC transporter MetNI exhibits both high-affinity transport toward l-methionine and broad specificity toward methionine derivatives, including d-methionine. In this work, we characterize the transport of d-methionine derivatives by the MetNI transporter. Unexpectedly, the N229A substrate-binding deficient variant of the cognate binding protein MetQ was found to support high MetNI transport activity toward d-selenomethionine. We determined the crystal structure at 2.95 Å resolution of the ATPγS-bound MetNIQ complex in the outward-facing conformation with the N229A apo MetQ variant. This structure revealed conformational changes in MetQ providing substrate access through the binding protein to the transmembrane translocation pathway. MetQ likely mediates uptake of methionine derivatives through two mechanisms: in the methionine-bound form delivering substrate from the periplasm to the transporter (the canonical mechanism) and in the apo form by facilitating ligand binding when complexed to the transporter (the noncanonical mechanism). This dual role for substrate-binding proteins is proposed to provide a kinetic strategy for ABC transporters to transport both high- and low-affinity substrates present in a physiological concentration range.

A15333 Relationship of Human CETP Deficiency Variant, HDL and Acute Myocardial Infarction in Chinese Population

A15333 Relationship of Human CETP Deficiency Variant, HDL and Acute Myocardial Infarction in Chinese Population

Relationship Between Vitamin D Status and Vitamin D Receptor Gene Polymorphisms With Markers of Metabolic Syndrome Among Adults

Introduction: Recent studies have demonstrated that vitamin D deficiency contributes to the development of metabolic disorders, including obesity and type 2 diabetes mellitus (T2DM). Several vitamin D receptor (VDR) gene polymorphisms had been described to play a role in these conditions since vitamin D receptors were found in many tissues. The aim of this study was to assess the relationship between vitamin D status and VDR gene polymorphisms with metabolic syndrome (MS) parameters in Russian middle-aged women.Materials and Methods: A total of 697 women aged between 30 to 55 years were included in this cross-sectional study. Serum 25-hydroxyvitamin D (25(OH)D) level and four VDR gene polymorphisms rs1544410 (BsmI), rs7975232 (ApaI), rs731236 (TaqI), and rs2228570 (FokI) were measured. We applied the International Diabetes Federation (IDF) criteria to identify subjects with MS.Results: 9.3% of subjects had normal vitamin D level, while 90.7% were insufficient or deficient. Abdominal obesity (AO) was seen in 75.5%, impaired glucose tolerance (IGT) or T2DM was observed in 33.3%, reduced high-density lipoprotein cholesterol (HDL-C) level in 32.2% and hypertriglyceridemia in 23.4%. Serum 25(OH)D level in women with or without MS did not differ (48.6 ± 1.8 and 51.1 ± 1.5 nmol/l, p > 0.05). Subjects with vitamin D deficiency showed an increased risk of AO [CI 95% 2.23; 1.15–4.30] and low HDL-C [CI95% 2.60; 1.04–6.49] compared to subjects with normal 25(OH)D level. IGT and T2DM risk was increased only when 25(OH)D concentration was less than 39.0 nmol/l [CI 95% 7.17; 2.99–17.7], but risk of MS did not differ in normal vitamin D status subjects and insufficient/deficient ones (p > 0.05). T allele carriers (A) of rs7975232 had higher total cholesterol and low-density lipoprotein cholesterol levels compared with the GG (aa) genotypes. Similarly, GG (BB) genotype carriers of rs1544410 had higher triglyceride levels than subjects with A (b) allele carriers. However VDR gene polymorphisms did not seem to be associated with an increased risk of MS.Conclusions: Vitamin D deficiency, rs7975232, and rs1544410 VDR gene variants are associated with MS parameters in Russian middle-aged women.