The interplay between SUCLA2, SUCLG2, and mitochondrial DNA depletion

SUCLA2-related mitochondrial DNA (mtDNA) depletion syndrome is a result of mutations in the β subunit of the ADP-dependent isoform of the Krebs cycle succinyl-CoA synthase (SCS). The mechanism of tissue specificity and mtDNA depletion is elusive but complementation by the GDP-dependent isoform encoded by SUCLG2, and the association with mitochondrial nucleoside diphosphate kinase (NDPK), is a plausible link.We have investigated this relationship by studying SUCLA2 deficient fibroblasts derived from patients and detected normal mtDNA content and normal NDPK activity. However, knockdown of SUCLG2 by shRNA in both patient and control fibroblasts resulted in a significant decrease in mtDNA amount, decreased NDPK and cytochrome c oxidase activities, and a marked growth impairment. This suggests that, SUCLG2, to a higher degree than SUCLA2, is crucial for mtDNA maintenance and that mitochondrial NDPK is involved. Although results pertain to a cell culture system, the findings might explain the pathomechanism and tissue specificity in mtDNA depletion caused by defective SUCLA2.     

Clinical,Molecular, and Computational Analysis in two cases with mitochondrial encephalomyopathy associated with SUCLG1 mutation in a consanguineous family

Deficiency of the mitochondrial enzyme succinyl COA ligase (SUCL) is associated with encephalomyopathic mtDNA depletion syndrome and methylmalonic aciduria. This disorder is caused by mutations in both SUCL subunits genes: SUCLG1 (α subnit) and SUCLA2 (β subnit). We report here, two Tunisian patients belonging to a consanguineous family with mitochondrial encephalomyopathy, hearing loss, lactic acidosis, hypotonia, psychomotor retardation and methylmalonic aciduria. Mutational analysis of SUCLG1 gene showed, for the first time, the presence of c.41T > C in the exon 1 at homozygous state. In-silico analysis revealed that this mutation substitutes a conserved methionine residue to a threonine at position 14 (p.M14T) located at the SUCLG1 protein mitochondrial targeting sequence. Moreover, these analysis predicted that this mutation alter stability structure and mitochondrial translocation of the protein. In Addition, a decrease in mtDNA copy number was revealed by real time PCR in the peripheral blood leukocytes in the two patients compared with controls.     

Pitfalls in screening streptococci for retrieving superior streptokinase (SK) genes: no activity correlation for streptococcal culture supernatant and recombinant SK

Streptokinase (SK), the heterogeneous protein family secreted by some groups of β-hemolytic streptococci (βHS), is a plasminogen activator and well-known drug for thrombolytic therapy. Differences in plasminogen activation property of streptococcal culture supernatants (SCS) have been traditionally used to identify superior producer strains and SK genes (skc) for recombinant SK (rSK) production. However, the role of SK heterogeneity and whether SK activities in SCS correlate with that of their corresponding rSK is a matter of debate. To address these concerns, SCS of nine group C streptococci (GCS) screened among 252 βHS clinical isolates were compared for plasminogen activation using S-2251 chromogenic assay. The GCS (Streptococcus equisimilis) showing the highest (GCS-S87) and lowest (GCS-S131) activities were selected for PCR-based isolation of skc, cloning and rSK production in Escherichia coli. The 6×His-tagged rSK proteins were purified by NI–NTA chromatography, analyzed by SDS-PAGE and Western blotting and their activities were determined. While SCS of GCS-S87 and GCS-S131 showed different plasminogen activations (95 and 35 %, respectively) compared to that of the reference strain (GCS-9542), but interestingly rSK of all three strains showed close specific activities (1.33, 1.70, and 1.55 × 10⁴ IU mg^?1). Accordingly, SKS87 and SKS131 had more than 90 % sequence identity at the amino acids level compared to SK9542. Therefore, SK heterogeneity by itself may not contribute to the differences in plasminogen activation properties of SCS and evaluation of this activity in SCS might not be a proper assay for screening superior skc.     

The Saccharomyces cerevisiae SCS2 Gene Product, a Homolog of a Synaptobrevin-Associated Protein, Is an Integral Membrane Protein of the Endoplasmic Reticulum and Is Required for Inositol Metabolism

The Saccharomyces cerevisiae SCS2 gene has been cloned as a suppressor of inositol auxotrophy of CSE1 and hac1/ire15 mutants (J. Nikawa, A. Murakami, E. Esumi, and K. Hosaka, J. Biochem. 118:39–45, 1995) and has homology with a synaptobrevin/VAMP-associated protein, VAP-33, cloned from Aplysia californica (P. A. Skehel, K. C. Martin, E. R. Kandel, and D. Bartsch, Science 269:1580–1583, 1995). In this study we have characterized an SCS2 gene product (Scs2p). The product has a molecular mass of 35 kDa and is C-terminally anchored to the endoplasmic reticulum, with the bulk of the protein located in the cytosol. The disruption of the SCS2 gene causes yeast cells to exhibit inositol auxotrophy at temperatures of above 34°C. Genetic studies reveal that the overexpression of the INO1 gene rescues the inositol auxotrophy of the SCS2 disruption strain. The significant primary structural feature of Scs2p is that the protein contains the 16-amino-acid sequence conserved in yeast and mammalian cells. The sequence is required for normal Scs2p function, because a mutant Scs2p that lacks the sequence does not complement the inositol auxotrophy of the SCS2 disruption strain. Therefore, the Scs2p function might be conserved among eukaryotic cells.     

Monomethylamine as a Nitrogen Source for a Nonmethylotrophic Bacterium,Agrobacterium tumefaciens

Monomethylamine can be used by nonmethylotrophs as a sole nitrogen source but not as a carbon source; however, little is known about the genes and enzymes involved. The γ-glutamylmethylamide/N-methylglutamate pathway for monomethylamine utilization by methylotrophs has recently been resolved. We have identified genes encoding key enzymes of this pathway in nonmethylotrophs (e.g., Agrobacterium tumefaciens) and demonstrated that this pathway is also involved in the utilization of monomethylamine as a nitrogen source by nonmethylotrophs.Monomethylamine (MMA) (CH₃NH₂) is ubiquitous in the environment and is released during the degradation of many nitrogen-containing compounds (1, 2, 6). Bacteria can use MMA as a sole carbon (C) and/or as a sole nitrogen (N) source (1). For methylotrophic bacteria that use MMA as a source of both C and N, different pathways have been elucidated, including the MMA dehydrogenase pathway and the MMA oxidase pathway and additional pathways involving methylated glutamate, i.e., γ-glutamylmethylamide (GMA) and N-methylglutamate (NMG) (1). The genes involved in the GMA/NMG-mediated MMA utilization pathway, including GMA synthetase (gmas), “NMG synthase” (mgsABC), and NMG dehydrogenase (mgdABCD), were recently identified in the methylotroph Methyloversatilis universalis (Fig. ​(Fig.11 A) (11). Although MMA can serve as a sole N source but not as a C source for many nonmethylotrophs (3, 5), the mechanisms involved are unclear. A search for GMA/NMG gene clusters in microbial genome sequence databases revealed that similar clusters are present in many nonmethylotrophs, including Agrobacterium tumefaciens C58, Rhizobium leguminosarum bv. viciae 3841, Mesorhizobium loti MAFF303099, and Ruegeria pomeroyi DSS-3. We tested the hypothesis that the GMA/NMG-mediated MMA utilization pathway is also involved in the metabolism of MMA as the sole N source by nonmethylotrophs.Open in a separate windowFIG. 1.(A) Gene organization of NMG dehydrogenase (mgdABCD), GMA synthetase (gmas), and “NMG synthase” (mgsABC) in representative methylotrophs and nonmethylotrophs. purU, formyltetrahydrofolate deformylase. (B) Proposed pathway of GMA- and NMG-mediated MMA metabolism in bacteria. The substrate specificity of “NMG synthase” is not well established (shown in dashed lines), and it is proposed that both MMA and GMA can be used as a substrate for this enzyme. MMA, monomethylamine; GMA, γ-glutamylmethylamide; NMG, N-methylglutamate; Glu, glutamate.     

The interplay between SUCLA2, SUCLG2, and mitochondrial DNA depletion

SUCLA2-related mitochondrial DNA (mtDNA) depletion syndrome is a result of mutations in the β subunit of the ADP-dependent isoform of the Krebs cycle succinyl-CoA synthase (SCS). The mechanism of tissue specificity and mtDNA depletion is elusive but complementation by the GDP-dependent isoform encoded by SUCLG2, and the association with mitochondrial nucleoside diphosphate kinase (NDPK), is a plausible link. We have investigated this relationship by studying SUCLA2 deficient fibroblasts derived from patients and detected normal mtDNA content and normal NDPK activity. However, knockdown of SUCLG2 by shRNA in both patient and control fibroblasts resulted in a significant decrease in mtDNA amount, decreased NDPK and cytochrome c oxidase activities, and a marked growth impairment. This suggests that, SUCLG2, to a higher degree than SUCLA2, is crucial for mtDNA maintenance and that mitochondrial NDPK is involved. Although results pertain to a cell culture system, the findings might explain the pathomechanism and tissue specificity in mtDNA depletion caused by defective SUCLA2.     

Association of PON1 and PON2 Polymorphisms with PON1 Activity and Significant Coronary Stenosis in a Tunisian Population

PON1 and PON2 have attracted considerable attention as candidate genes for coronary heart disease because their enzymes function as key factors in lipoprotein catabolism pathways. We studied the distribution of PON1 and PON2 polymorphisms, including genotyping, lipid profile, and PON1 activity, and their association with PON1 activity and significant coronary stenosis (SCS) in a Tunisian population. PON1 activity was lower in patients with SCS than in controls. It increased with the R allele (QQ < QR < RR) in PON1-192 genotypes and with the L allele (MM < ML < LL) in PON1-55 genotypes. In the presence of metabolic syndrome and diabetes, PON1-192RR and PON2-311CC were associated with an increased risk of SCS and PON1-55MM seems to have lower risk. This association was evident among nonsmokers for PON1-55MM and among smokers for PON1-192RR and PON2-311CC. The GTGC haplotype seemed to increase the risk of SCS compared with the wild haplotype in a Tunisian population.     

Marked mitochondrial DNA depletion associated with a novel SUCLG1 gene mutation resulting in lethal neonatal acidosis,multi-organ failure,and interrupted aortic arch

The aim of this study was to identify the causative genetic lesion in two apparently unrelated newborns having lethal lactic acidosis, multi-organ failure and congenital malformations including interrupted aortic arch, who exhibited mild methylmalonic aciduria, combined mitochondrial respiratory chain deficiency, and marked muscle mitochondrial DNA depletion. A novel mutation in the SUCLG1 gene was identified. Phenotype severity in Succinate-CoA ligase dysfunction appears to be more correlated to the muscle mtDNA content than to the tissue distribution of the heterodimer subunits. Prominent impairment of mitochondrial respiratory chain may result in deep ravages in developmental tissues leading to multiple organ failure and malformations.     

The ASCT/SCS cycle fuels mitochondrial ATP and acetate production in Trypanosoma brucei

Acetate:succinate CoA transferase (ASCT) is a mitochondrial enzyme that catalyzes the production of acetate and succinyl-CoA, which is coupled to ATP production with succinyl-CoA synthetase (SCS) in a process called the ASCT/SCS cycle. This cycle has been studied in Trypanosoma brucei (T. brucei), a pathogen of African sleeping sickness, and is involved in (i) ATP and (ii) acetate production and proceeds independent of oxygen and an electrochemical gradient. Interestingly, knockout of ASCT in procyclic form (PCF) of T. brucei cause oligomycin A-hypersensitivity phenotype indicating that ASCT/SCS cycle complements the deficiency of ATP synthase activity. In bloodstream form (BSF) of T. brucei, ATP synthase works in reverse to maintain the electrochemical gradient by hydrolyzing ATP. However, no information has been available on the source of ATP, although ASCT/SCS cycle could be a potential candidate. Regarding mitochondrial acetate production, which is essential for fatty acid biosynthesis and growth of T. brucei, ASCT or acetyl-CoA hydrolase (ACH) are known to be its source. Despite the importance of this cycle, direct evidence of its function is lacking, and there are no comprehensive biochemical or structural biology studies reported so far. Here, we show that in vitro–reconstituted ASCT/SCS cycle is highly specific towards acetyl-CoA and has a higher k_cat than that of yeast and bacterial ATP synthases. Our results provide the first biochemical basis for (i) rescue of ATP synthase-deficient phenotype by ASCT/SCS cycle in PCF and (ii) a potential source of ATP for the reverse reaction of ATP synthase in BSF.     

Methylmalonic acid administration induces DNA damage in rat brain and kidney

Accumulation of methylmalonic acid (MMA) in tissues and biological fluids is the biochemical hallmark of methylmalonic aciduria. Affected patients present renal failure and severe neurological findings. Considering that the underlying pathomechanisms of tissue damage are not yet understood, in the present work we assessed the in vivo e in vitro effects of MMA on DNA damage in brain and kidney, as well as on p53 and caspase 3 levels, in the presence or absence of gentamicin (acute renal failure model). For in vitro studies, tissue prisms were incubated in the presence of different concentrations of MMA and/or gentamicin for one hour. For in vivo studies, animals received a single injection of gentamicin (70 mg/kg) and/or three injections of MMA (1.67 μmol/g; 11 h interval between injections). The animals were killed 1 h after the last MMA injection. Controls received saline in the same volumes. DNA damage was analyzed by the comet assay. We found that MMA and gentamicin alone or combined in vitro increased DNA damage in cerebral cortex and kidney of rats. Furthermore, MMA administration increased DNA damage in both brain and kidney. Gentamicin per se induced DNA damage only in kidney, and the association of MMA plus gentamicin also caused DNA damage in cerebral cortex and kidney. On the other hand, p53 and caspase 3 levels were not altered by the administration of MMA and/or gentamicin. Our findings provide evidence that DNA damage may contribute to the neurological and renal damage found in patients affected by methylmalonic aciduria.     

The Rice Aquaporin Lsi1 Mediates Uptake of Methylated Arsenic Species

Pentavalent methylated arsenic (As) species such as monomethylarsonic acid [MMA(V)] and dimethylarsinic acid [DMA(V)] are used as herbicides or pesticides, and can also be synthesized by soil microorganisms or algae through As methylation. The mechanism of MMA(V) and DMA(V) uptake remains unknown. Recent studies have shown that arsenite is taken up by rice (Oryza sativa) roots through two silicon transporters, Lsi1 (the aquaporin NIP2;1) and Lsi2 (an efflux carrier). Here we investigated whether these two transporters also mediate the uptake of MMA(V) and DMA(V). MMA(V) was partly reduced to trivalent MMA(III) in rice roots, but only MMA(V) was translocated to shoots. DMA(V) was stable in plants. The rice lsi1 mutant lost about 80% and 50% of the uptake capacity for MMA(V) and DMA(V), respectively, compared with the wild-type rice, whereas Lsi2 mutation had little effect. The short-term uptake kinetics of MMA(V) can be described by a Michaelis-Menten plus linear model, with the wild type having 3.5-fold higher V_max than the lsi1 mutant. The uptake kinetics of DMA(V) were linear with the slope being 2.8-fold higher in the wild type than the lsi1 mutant. Heterologous expression of Lsi1 in Xenopus laevis oocytes significantly increased the uptake of MMA(V) but not DMA(V), possibly because of a very limited uptake of the latter. Uptake of MMA(V) and DMA(V) by wild-type rice was increased as the pH of the medium decreased, consistent with an increasing proportion of the undissociated species. The results demonstrate that Lsi1 mediates the uptake of undissociated methylated As in rice roots.Arsenic (As) contamination affects millions of people worldwide, particularly in South Asia where As-contaminated groundwater has been extracted for drinking (Chakraborti et al., 2002; Nordstrom, 2002). Recent studies have shown that foods, especially rice (Oryza sativa), are an important source of inorganic As to populations dependent on a rice diet (Kile et al., 2007; Ohno et al., 2007; Mondal and Polya, 2008). Paddy rice is more efficient than other cereal crops in accumulating As (Williams et al., 2007). This is because anaerobic conditions in submerged paddy soils lead to mobilization of arsenite [As(III); Takahashi et al., 2004; Xu et al., 2008], which is then taken up by rice roots mainly through the highly efficient transport pathway for silicon (Si; Ma et al., 2008). The relatively high accumulation of As in rice is of concern, as it may pose a significant health risk (Zhu et al., 2008; Meharg et al., 2009).A number of As species may be present in soil depending on soil conditions and the history of As contamination. These include arsenate [As(V)], As(III), and methylated As species such as monomethylarsonic acid [MMA(V): CH₃AsO(OH)₂] and dimethylarsinic acid [DMA(V): (CH₃)₂AsO(OH)]. As(V) is the main species in aerobic soils, while As(III) dominates in anaerobic environments such as flooded paddy soils. Both MMA(V) and DMA(V) have been found in paddy soils (Takamatsu et al., 1982), which may have been derived from microbial and algal biomethylation and/or past uses of methylated As compounds. MMA(V), as sodium or calcium salt, and DMA(V), as sodium salt or free acid (also called cacodylic acid), are herbicides widely used for weed control on cotton (Gossypium hirsutum), orchards, and lawns, or as a defoliant of cotton (U.S. Environmental Protection Agency, 2006). Conversion of cotton fields for the production of paddy rice in the United States may be a reason for the high levels of methylated As reported for the U.S. rice (Meharg et al., 2009).The mechanism of As(V) uptake by plants through the phosphate transport system has been well established (for review, see Zhao et al., 2009). In contrast, As(III) is taken up into the cells by aquaglyceroporins in Escherichia coli, yeast (Saccharomyces cerevisiae), and mammalian tissues (for review, see Bhattacharjee and Rosen, 2007). Recent studies have shown that several plant aquaporin channels belonging to the Nodulin 26-like Intrinsic Protein (NIP) subfamily are permeable to As(III) when expressed heterologously in yeast (Bienert et al., 2008; Isayenkov and Maathuis, 2008; Ma et al., 2008). The rice Si transporter Lsi1 (OsNIP2;1; Ma et al., 2006) is also permeable to As(III) when expressed in yeast or Xenopus laevis oocytes (Ma et al., 2008). Furthermore, the lsi1 rice mutant lost 57% of the influx capacity for As(III) compared to the wild type in short-term assays, suggesting that Lsi1 is an important entry route for As(III) (Ma et al., 2008). In rice roots, a second Si transporter, Lsi2, functions as an efflux carrier to transport Si efflux from the exodermis and endodermis cells toward the stele for xylem loading (Ma et al., 2007). This transporter also mediates As(III) efflux; two independent lsi2 mutants had 73% to 91% lower concentrations of As(III) in the xylem sap than their wild types (Ma et al., 2008). The shared uptake pathway between Si (silicic acid) and As(III) (arsenous acid) is consistent with their physiochemical properties; both are present predominantly as undissociated neutral molecules at the normal environmental and physiological pH range (pK_a = 9.2, >99% undissociated at pH ≤ 7.0), and the two molecules have similar sizes.The uptake mechanisms of methylated As species by plant roots are not known. Previous studies showed that both MMA(V) and DMA(V) can be taken up by roots and translocated to shoots in a number of plant species (Marin et al., 1992; Carbonell-Barrachina et al., 1998, 1999; Burló et al., 1999). Marin et al. (1992) found that uptake by rice followed the order of As(III) > MMA(V) > As(V) > DMA (V), although DMA(V) was more efficiently translocated from roots to shoots than other As species. Raab et al. (2007) reported large variations in the absorption and translocation efficiencies for As(V), MMA(V), and DMA(V) among the 46 plant species tested. On average, root absorption of As(V) was 2.5- and 5-times higher than MMA(V) and DMA(V), respectively. The translocation efficiency, defined as the shoot-to-root concentration ratio after 24-h exposure, was highest for DMA(V) (0.8), followed by MMA(V) (0.3) and As(V) (0.09). The concentration-dependent uptake kinetics of MMA(V) in rice roots could be described by the Michaelis-Menten equation, whereas the limited uptake of DMA(V) appeared to be linear in relation to the increasing concentration in the uptake medium (Abedin et al., 2002). Abbas and Meharg (2008) showed that DMA(V) uptake by maize (Zea mays) seedlings was enhanced by more than 10-fold by a pretreatment of phosphorus starvation; this compared with only 2-fold increase in As(V) uptake. They thought that DMA(V) might be taken up by the phosphate transporters, or that phosphorus starvation altered expression of a range of membrane transporters or even membrane permeability itself.In addition to the root uptake of methylated As species, some plants appear to be able to biomethylate As, but the pathway and enzymology remains unclear (Wu et al., 2002; Zhao et al., 2009). In microbes, As methylation follows the Challenger pathway involving repeated steps of As reduction and oxidative methylation (Bentley and Chasteen, 2002). As(V) is first reduced to As(III), which is methylated by S-adenosylmethyltransferase using S-adenosyl-l-Met as the methyl donor. The product of this reaction is pentavalent MMA(V), which is reduced by a reductase to trivalent MMA(III) with thiols (e.g. glutathione). Methylation and reduction steps continue to produce di- and trimethyl As compounds. MMA(III) and DMA(III) are intermediates in the As methylation pathway, which is not very stable (Gong et al., 2001). In rice grain, DMA(V) is the main form of methylated As, and can account for up to 80% of the total As (Zavala et al., 2008; Meharg et al., 2009). In light of the significant presence of methylated As in rice, it is important to elucidate the transport and assimilation pathways of these As species in plants.In this study, we present evidence that MMA(V) and DMA(V) are taken up by rice roots, at least partly, through the NIP aquaporin channel Lsi1, and that this process is strongly pH dependent. We also show that MMA(V) can be reduced to MMA(III) in planta.     

Serum vitamin B12 not reflecting vitamin B12 status in patients with type 2 diabetes

Contradictory results for concentrations of vitamin B12, holotranscobalamin (holoTC), and methylmalonic acid (MMA) have been reported. We tested the hypothesis that the extracellular vitamin B12 markers are not reflecting the intracellular vitamin B12-dependent biochemical reactions in individuals with type 2 diabetes. The study included 92 patients with diabetes and 72 controls with similar age and sex distribution. We measured vitamin B12 markers [MMA, total serum vitamin B12, holoTC, total homocysteine (tHcy)], red blood cell (RBC)-B12, and the plasma concentrations of the methylation markers [S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH)]. In comparison to controls, diabetic patients showed significantly higher concentrations of plasma SAH (median 15.1 vs. 11.8 nmol/L; p < 0.001) and lower SAM/SAH ratio (9.1 vs. 8.2; p = 0.006). Concentrations of total vitamin B12 and holoTC did not differ significantly between the groups, but plasma MMA concentrations were significantly higher in diabetics (250 vs. 206 nmol/L). However, RBC-B12 was lower in diabetics compared to controls (median 230 vs. 260 pmol/L; p = 0.001). The inverse correlation between MMA and RBC-B12 was stronger in the controls compared to that in the patients (correlation coefficient in controls R = −0.446, p = 0.001; in patients R = −0.289, p = 0.022). Metformin treatment was associated with a lower total serum vitamin B12, but a comparable RBC-B12 and a slightly lower MMA and better methylation index. In conclusion, patients with type 2 diabetes showed normal extracellular vitamin B12, but disturbed intracellular B12-dependent biochemical reactions. Metformin treatment was associated with low serum vitamin B12 and improved intracellular vitamin B12 metabolism despite low serum vitamin B12.     

Collated mutations in mitochondrial DNA (mtDNA) depletion syndrome (excluding the mitochondrial gamma polymerase,POLG1)

These tables list both published and a number of unpublished mutations in genes associated with early onset defects in mitochondrial DNA (mtDNA) maintenance including C10orf2, SUCLG1, SUCLA2, TYMP, RRM2B, MPV17, DGUOK and TK2. The list should not be taken as evidence that any particular mutation is pathogenic. We have included genes known to cause mtDNA depletion, excluding POLG1, because of the existing database (http://tools.niehs.nih.gov/polg/). We have also excluded mutations in C10orf2 associated with dominant adult onset disorders.     

Absence of MMACHC in peripheral retinal cells does not lead to an ocular phenotype in mice

Combined methylmalonic aciduria with homocystinuria (cblC type) is a rare disease caused by mutations in the MMACHC gene. MMACHC encodes an enzyme crucial for intracellular vitamin B₁₂ metabolism, leading to the accumulation of toxic metabolites e.g. methylmalonic acid (MMA) and homocysteine (Hcy), and secondary disturbances in folate and one-carbon metabolism when not fully functional. Patients with cblC deficiency often present in the neonatal or early childhood period with a severe multisystem pathology, which comprises a broad spectrum of treatment-resistant ophthalmological phenotypes, including retinal degeneration, impaired vision, and vascular changes. To examine the potential function of MMACHC in the retina and how its loss may impact disease, we performed gene expression studies in human and mouse, which showed that local expression of MMACHC in the retina and retinal pigment epithelium is relatively stable over time. To study whether functional MMACHC is required for retinal function and tissue integrity, we generated a transgenic mouse lacking Mmachc expression in cells of the peripheral retina. Characterization of this mouse revealed accumulation of cblC disease related metabolites, including MMA and the folate-dependent purine synthesis intermediates AICA-riboside and SAICA-riboside in the retina. Nevertheless, fundus appearance, morphology, vasculature, and cellular composition of the retina, as well as ocular function, remained normal in mice up to 6 or 12 months of age. Our data indicates that peripheral retinal neurons do not require intrinsic expression of Mmachc for survival and function and questions whether a local MMACHC deficiency is responsible for the retinal phenotypes in patients.     

Spatial variations in archaeal lipids of surface water and core-top sediments in the South china sea and their implications for paleoclimate studies

The South China Sea (SCS) is the largest marginal sea of the western Pacific Ocean, yet little is known about archaeal distributions and TEX₈₆-based temperatures in this unique oceanic setting. Here we report findings of abundances in both core lipids (CL) and intact polar lipids (IPL) of Archaea from surface water (CL only) and core-top sediments from different regions of the SCS. TEX₈₆-derived temperatures were also calculated for these samples. The surface water had extremely low abundances of CL (average of 0.05 ± 0.13 ng/liter; n = 75), with higher values present in regions where upwelling is known to occur. The core-top sediments had CL values of 0.1 to 0.9 μg/g, which are on the low end of CL concentrations reported for other marine sediments and may reflect the oligotrophic nature of the open SCS. The IPL of Archaea accounted for 6 to 36.4% of total lipids (CL plus IPL), indicating that the majority of archaeal lipids in core-top sediments were derived from nonliving cells. The TEX₈₆-based temperatures of surface water were overall lower than satellite-based sea surface temperatures or CTD-measured in situ temperatures. The core-top sediment samples, however, had TEX₈₆ temperatures very close to the mean annual sea surface temperatures, except for samples with water depths of less than 100 m. Our results demonstrated low and heterogeneous distributions of archaeal lipids in surface water and core-top sediments of the SCS, which may reflect local or regional differences in productivity of Archaea. While TEX₈₆-based temperatures for core-top marine sediments at deep water depths (>100 m) generally reflected mean annual sea surface temperatures, TEX₈₆ temperatures in surface water varied basin wide and underestimated sea surface temperatures in most locations for the season when surface water samples were collected.     

Exceptions in patterns of arsenic compounds in urine of acute promyelocytic leukaemia patients treated with As<Subscript>2</Subscript>O<Subscript>3</Subscript>

Arsenic trioxide (As(III) in solution) has been shown to be the most active single agent in combating acute promyelocytic leukemia (APL). It is metabolized and excreted via urine as monomethylarsonic acid (MMA), dimethylarsinic acid (DMA) and As(V), along with excess As(III). In our study eight APL patients were treated (intravenously) with 0.15 mg As₂O₃/kg/day. During the therapy As(III) and its metabolites were followed in pre- and post-infusion urine using HPLC for separation followed by on-line detection using hydride generation-atomic fluorescence spectrometry. Five patients had a normal excretion pattern of residual arsenic compounds in morning pre-infusion urine, with 15–25 % of As(III), 35–55 % of DMA, 25–30 % of MMA and 1–5 % of As(V), while three patients showed unexpected exceptions from typical excretion patterns of arsenic compounds (i) a high DMA/MMA ratio (factor 5.3), (ii) severe As(III) oxidation (10.2 % As(III) converted to As(V)) or (iii) the presence of an excessive amount of As(III) (average 30.4 % of total arsenic). Intriguing was the occurrence of post-infusion oxidation of As(III) to As(V) observed in almost all patients and being especially high (>40 %) in patient with increased residual As(V). Results indicate that arsenic metabolites patterns can be unpredictable. Observed high levels of un-metabolised As(III) are a warning signal for side effects and for routine determination of arsenic metabolites during first days of treatment. High or low percentages of MMA or DMA did not show any observable effect on treatment results, while clear presence of post-infusion As(V) supports theoretical claims of in vivo oxidation (detoxification) of As(III) to As(V) associated with various metabolic processes.