GLO Activity Research Articles

Ascorbic acid biosynthesis in Pacific abalone Haliotis discus hannai Ino and L-gulonolactone oxidase gene loss as an independent event

Up to now, it has been believed that invertebrates are unable to synthesize ascorbic acid (AA) in vivo. However, in the present study, the full-length CDs (Coding sequence) of L-gulonolactone oxidase (GLO) from Pacific abalone (Haliotis discus hannai Ino) were obtained through molecular cloning. The Pacific abalone GLO contained a FAD-binding domain in the N-termination, and ALO domain and conserved HWAK motif in the C-termination. The GLO gene possesses 12 exons and 11 introns. The Pacific abalone GLO was expressed in various tissues, including the kidney, digestive gland, gill, intestine, muscle and mantle. The GLO activity assay revealed that GLO activity was only detected in the kidney of Pacific abalone. After a 100-day feeding trial, dietary AA levels did not significantly affect the survival, weight gain, daily increment in shell length, and feed conversion ratio of Pacific abalone. The expression of GLO in the kidney was downregulated by dietary AA. These results implied that the ability to synthesize AA in abalone had not been lost. From the evolutionary perspective, the loss of GLO occurred independently as an independent event by matching with the genomes of various species. The positive selection analysis revealed that the GLO gene underwent purifying selective pressure during its evolution. In conclusion, the present study provided direct evidence to prove that the GLO activity and the ability to synthesize AA exist in abalone. The AA synthesis ability in vertebrates might have originated from invertebrates dating back 930.31 million years.

Catalytic and functional aspects of different isozymes of glycolate oxidase in rice

BackgroundGlycolate oxidase (GLO) is a key enzyme for photorespiration in plants. There are four GLO genes encoding and forming different isozymes in rice, but their functional differences are not well understood. In this study, enzymatic and physiological characteristics of the GLO isozymes were comparatively analyzed.ResultsWhen expressed heterologously in yeast, GLO1, GLO4 and GLO1 + 4 showed the highest activities and lowest Km for glycolate as substrate, whereas GLO3 displayed high activities and affinities for both glycolate and L-lactate, and GLO5 was catalytically inactive with all substrates tested. To further reveal the physiological role of each GLO isozyme in plants, various GLO genetically modified rice lines were generated and functionally analyzed. GLO activity was significantly increased both in GLO1 and GLO4 overexpression lines. Nevertheless, when either GLO1 or GLO4 was knocked out, the activity was suppressed much more significantly in GLO1 knockout lines than in GLO4 knockout lines, and both knockout mutants exhibited obvious dwarfism phenotypes. Among GLO3 and GLO5 overexpression lines and RNAi lines, only GLO3 overexpression lines showed significantly increased L-lactate-oxidizing activity but no other noticeable phenotype changes.ConclusionsThese results indicate that rice GLO isozymes have distinct enzymatic characteristics, and they may have different physiological functions in rice.

Ascorbic acid regulation in stress responses during acute cold exposure and following recovery in juvenile Chinese soft-shelled turtle (Pelodiscus sinensis)

Intense temperature change often leads to increased oxidative stress in many animals with a few exceptions, including the turtle. To date, little is known about the mechanism of protective antioxidative defenses in turtles during acute temperature change, specifically the role that the antioxidant ascorbic acid (AA) plays. In this study, Chinese soft-shelled turtles (Pelodiscus sinensis) were initially acclimated at 28°C (3wks), exposed to acute cold condition (8°C, 8h) and finally placed in recovery (28°C, 24h). L-Gulonolactone oxidase (GLO) mRNA exhibited a stable transcription pattern during the intense thermal fluctuation. GLO activity also remained stable, which validated the mRNA expression pattern. The similar Q10 values for GLO activity in the different treatment groups at incubation temperatures of 28°C and 8°C indicated that the GLO activity response to thermal change exhibited a temperature-dependent enzymatic kinetic characteristic. The AA storage was tissue-specific as well as the AA re-supply in the recovery period, with brain as the priority. Despite the insufficient transport during cold exposure, the plasma AA reservoir greatly contributed to the redistribution of AA during recovery. Depending on the prominent GLO activity, the high level of tissue-specific AA storage and the extraordinary plasma AA transport potential, the Chinese soft-shelled turtle endured severe thermal fluctuations with no apparent oxidative stress. However, the significant decrease in AA concentration in the brain tissue during acute cold exposure suggested that such a strategy may not be sufficient for prolonged cold exposure.

Effect of chronic oxidative/corticosterone‐induced stress on ascorbic acid metabolism and total antioxidant capacity in chickens (Gallus gallus domesticus)

The consequences of chronic corticosterone-induced stress (CCIS) on ascorbic acid (AsA) metabolism in chickens, an animal that syntheses the vitamin, are not known. This study was conducted to determine whether CCIS alters AsA synthesis, as measured by l-gulonolactone oxidase (GLO) activity, tissue AsA, lipid peroxides and tissue total antioxidant capacity (TAC). Stress was induced by dietary administration of corticosterone from 2 to 4 weeks of age and measurements were made at 0, 7 and 14 days post-treatment. Ascorbic acid synthesis was not influenced by CCIS but hepatic, cardiac, renal, bursal and duodenal AsA concentrations were significantly decreased and plasma TAC and uric acid concentrations were significantly elevated. Stress caused significant hepatomegaly and hepatic lipidosis but hepatic peroxides were not elevated despite the slight decrease in hepatic TAC. Tissue TAC varied in different organs. It was markedly elevated in the kidney, reduced by 49% in the spleen, and changes were not detected in the heart and duodenum even though AsA concentration was significantly decreased in all tissues. We conclude that CCIS caused a significant reduction in tissue AsA concentration but did not inhibit GLO activity. The change in AsA concentration was associated with increase, decrease or no change in TAC in tissues examined. The findings suggest that CCIS may alter AsA recycling, influx or turnover in different tissues of chickens.

Developmental aspects and factors influencing the synthesis and status of ascorbic Acid in the pig.

Ascorbic acid synthesis in the pig occurs at mid-pregnancy, but activity of the enzyme l-gulono-gamma-lactone oxidase (GLO) declines thereafter during gestation and remains low when the pig nurses the sow. During late gestation the ascorbic acid concentration in the fetus increases, but serum and liver ascorbic acid concentration in the sow declines without affecting the dam's liver GLO activity. It is presumed that as gestation progresses an increased amount of maternal ascorbic acid is transferred to the fetus and to the mammary gland. Colostrum and milk are rich sources of the vitamin and supply the nursing pig with ascorbic acid. The available data suggest that high amounts of ascorbic acid appear to suppress liver GLO activity in the pig. Upon weaning, when exogenous vitamin C is generally not provided, liver GLO activity and serum ascorbic acid increases. During the initial periods postweaning, some reports have indicated growth benefits of supplemental vitamin C. Body tissues differ in their concentrations of ascorbic acid, but tissues of high metabolic need generally have greater concentrations. The corpus luteum in the female, the testis in the male, and the adrenal glands in all pigs contain greater concentrations of the vitamin. Knockout genes preventing ascorbic acid synthesis in pigs have demonstrated poor skeletal and collagen formation and poor antioxidant protection. Under periods of stress ascorbic acid declines in the adrenal, but the pig rapidly recovers to its resting state once the stressor agent is removed. Although there are periods when supplemental vitamin C has been shown to promote pig performance (e.g., during high environmental stress and early postweaning), supplemental vitamin C has not been shown to routinely enhance pig performance.

Factors affecting ascorbic acid biosynthesis in chickens: III. Effect of dietary fluoride on L-gulonolactone oxidase activity and tissue ascorbic acid (AsA) concentration.

The inconsistent beneficial responses to dietary ascorbic acid (AsA) may be due to dietary factors that alter biosynthesis or tissue turnover of AsA. It has been suggested on the basis of altered tissue AsA that dietary fluoride is a determinant of biosynthesis in chickens. Fluoride may enter the food chain of poultry via industrial contamination, feed ingredients and drinking water. The goal of this study was to ascertain whether dietary fluoride at 300 mg/kg influences l-gulonolactone oxidase (GLO) activity in commercial meat-type chickens. The experimental diet was fed from day-old to 3 weeks and responses measured. Growth and feed conversion were not affected by fluoride in the diet. Dietary fluoride neither inhibited nor enhanced GLO activity nor did it increase or decrease AsA concentration in plasma, liver, kidney, adrenal gland and muscle (pectoralis major). Tissue AsA concentration in ascending order was adrenal > liver > kidney > pectoralis major > plasma. The results are consistent with that reported for the rat and calculations based on the results eliminate fluorine contamination for the inconsistent responses of immature chickens to dietary AsA.

Factors affecting ascorbic acid (AsA) biosynthesis in chickens. II. Effect of dietary AsA and strain of chicken

The present study examined the effect of supplemental ascorbic acid (AsA) and ascertained if genotype is a determinant of biosynthesis and the response of strains to dietary AsA. Slow- (Ottawa Meat Control; OMC) and fast-growing (Peterson Enhanced x Hubbard; PEH) chicks were fed 1000 mg/kg AsA from 1 to 10 weeks of age. The activity of l-gulonolactone oxidase (GLO) was used to measure biosynthesis and estimated synthetic capacity (ESC) computed. Body weight was not affected by diets and relative kidney weight decreased with age. In 1 week, dietary AsA increased plasma AsA and inhibited GLO activity with a greater reduction in OMC birds. At 10 weeks, GLO activity was depressed almost uniformly in both strains. Strain by age and diet by age interactions were detected for GLO activity and ESC with significantly greater decline in PEH birds and birds fed supplemental AsA. The results demonstrated that dietary AsA inhibited biosynthesis in meat type chickens and the response at 10 weeks was not influenced by growth rate; and the age dependent decline in biosynthesis was more pronounced in the commercial PEH birds. The result suggests that such strains may be compromised in some situations. Research using multiple dietary levels of AsA, commercial strains, and defined stressors is warranted.

Isolation and transient expression of a cDNA encoding l-gulono-γ-lactone oxidase, a key enzyme for l-ascorbic acid biosynthesis, from the tiger shark Scyliorhinustorazame

We present the first description of a cDNA encoding the l-gulono-γ-lactone oxidase (GLO) from a fish, a cartilaginous shark species, Scyliorhinus torazame. An expressed sequence tag (EST) from the shark kidney, which showed high similarity with a rat GLO gene, was isolated and its full-length sequence (1752 bp) was determined. The putative shark GLO (sGLO) cDNA sequence contained 98 bp of 5′-untranslated region, an open reading frame consisting 440 amino acids, and 334 bp of 3′-untranslated region including the poly(A+) tail. The deduced amino acid sequence was 63% identical to the rat GLO sequence and showed high conservation in the flavine adenine dinucleotide (FAD)-binding region. In addition, the calculated molecular mass (50,976 Da), theoretical p I (7.17) and hydropathy profile were similar to those of the rat GLO. Using both reverse transcription-PCR (RT-PCR) assays and the sGLO cDNA as a probe in Northern hybridisation experiments, expression was demonstrated in the shark kidney but not in any other tissues (brain, intestine, liver, muscle, pituitary and spleen). Biologically functional GLO activity was demonstrated by direct delivery of an expression vector containing the sGLO cDNA into kidney of far eastern catfish ( Silurus asotus), which lacks endogenous GLO activity. Transient expression of GLO activity was dependent on the amount of plasmid injected (up to 120 μg of DNA), and persisted for 12 days post injection, as demonstrated by RT-PCR and biochemical assays.

Ascorbic Acid Synthesis in Fetal and Neonatal Pigs and in Pregnant and Postpartum Sows

The ontogeny of ascorbic acid synthesis and its concentration in fetal pigs from mid- to late gestation, and the effect of birth order and premature or normal delivery ages were evaluated. In Experiment 1, fetal pigs were collected from three sows at 60, 80, 100, 107 and 111 d of development. Liver L-gulono-gamma-lactone oxidase (GLO) activity and ascorbic acid concentration were measured. High liver GLO activity in fetal liver occurred at 60 d but declined as pregnancy advanced (P < 0.01), whereas ascorbic acid concentration increased (P < 0.01). Experiment 2 evaluated ascorbic acid synthesis and concentration in neonates born early (1st and 2nd) or late (7th and 8th) in the birthing sequence, or when born 2 d prematurely vs. the normal delivery age. Pigs born early in the birthing sequence (P < 0.01) and those born at the natural delivery age (P < 0.05) had higher liver ascorbic acid concentrations, but liver GLO activity did not differ among groups. Sows were killed at each period; liver GLO activity was constant during gestation but increased postpartum (P < 0.01). Liver ascorbic acid concentration was constant during gestation, except for a decline during late gestation, and increased postpartum (P < 0.05). These results suggest that more ascorbic acid was transferred from the dam to the fetuses as pregnancy advanced, possibly suppressing fetal GLO activity. Thus, fetal liver GLO activity was the primary source of ascorbic acid during early fetal development, but more fetal ascorbic acid was transferred from the dam during later pregnancy.

Liver L-Gulonolactone Oxidase Activity and Tissue Ascorbic Acid Concentrations in Nursing Pigs and the Effect of Various Weaning Ages

In Experiment 1, we evaluated liver L-gulono-gamma-lactone oxidase (GLO) activity and tissue concentration of ascorbic acid in young pigs from birth to weaning (14 d) and through a 28-d postweaning period; in Experiment 2, we evaluated the effect of three weaning ages on these measurements. Sow colostrum and milk collected in both experiments demonstrated a linear decline (P < 0.01) in ascorbic acid concentration as lactation progressed. In Experiment 1, three pigs were killed at 0, 3, 7, 14, 21, 28, 35 and 42 d of age for determining liver GLO activity and serum and tissue ascorbic acid. Liver GLO activity decreased by 80% from 0 to 3 d of age and remained low until d 14 (weaning). After weaning, liver GLO activity increased linearly (P < 0.01). Tissue ascorbic acid concentrations decreased during the nursing period and again after weaning, but then increased to 42 d of age (P < 0.01). In Experiment 2, pigs were weaned at 10, 17 or 24 d of age. Three pigs from each group were killed at weaning and at each week postweaning until 38 d of age. Liver GLO activity was low during the nursing period but increased linearly (P < 0.01) for each group during the subsequent postweaning period. Pig serum and tissue ascorbic acid concentrations increased postweaning in each group. These results suggest that a factor in sow's milk, possibly ascorbic acid, suppressed liver GLO activity of nursing pigs but upon weaning, liver GLO activity of pigs increased in a linear manner (P < 0.01).

Ascorbic acid biosynthesis in Amazonian fishes

The incapacity to synthesize ascorbic acid (AA) is due to the lack of activity of l-gulonolactone oxidase (GLO), which catalyzes the last step of AA biosynthesis. It was postulated that vertebrates unable to synthesize AA had sufficient amounts of this nutrient in their diet and consequently did not need to preserve synthetic capability. In the present study, we analyzed the GLO activity in kidney and liver of 13 fish species, including 11 teleosts, namely: freshwater stingray, Potamotrygon sp.; South American lungfish, Lepidosiren paradoxa; “sardinhão,” Pelona sp.; arowana, Osteoglossum bicirrhosum; arapaima, Arapaima gigas; “piranha caju,” Pygocentrus nattereri; “piranha mucura,” Serrasalmus elongatus; “aracu,” Schizodon fasciatus; “tambaqui,” Colossoma macropomum; “acari-pedra,” Hypostomus sp.; “sarapó,” Steatogenys elegans; electric eel, Electrophorus electricus; and the peacock bass, Cichla sp. Four representatives of the Characiformes order with distinct feeding habits were included in this study to evaluate the influence of feeding habit on GLO activity. Only two species of non-teleost fishes, the freshwater stingray (Miliobatiformes) and the South American lungfish (Lepidosireniformes), showed GLO activity in their kidneys, corroborating the hypothesis that teleosts are unable to synthesize AA. Additionally, as expected, we observed that the phylogenetic position is more important than feeding habit as a determinant of the biosynthetic ability since none of the Characiformes species analyzed synthesize AA, independent of their distinct feeding habits. Female freshwater stingrays had a significantly higher GLO activity than males ( P<0.05), but there was no difference in the biosynthetic capability regarding the portion of the kidney analyzed ( P>0.05), as previously reported in white sturgeon. This is the first report of the AA biosynthetic ability in South American lungfish and freshwater Elasmobranchs.

Factors affecting ascorbic acid biosynthesis in chickens. I. Adaptation of an assay and the effect of age, sex and food deprivation

SummaryThe objective of this study was to adapt an assay to measure L‐gulonolactone oxidase (GLO) activity, the final step in ascorbate synthesis, in chickens and use the procedure to study the effect of gender, age, and food deprivation. To adapt a procedure used for mammalian tissues the kinetic constants were estimated using chicken kidney and assay conditions optimized in terms of storage of tissue and homogeneity of distribution. A series of experiments were conducted using the standardized assay with individual animals as the experimental unit. Gender differences were detected only in mature birds with male chickens having lower GLO activity than females. Time‐course changes in GLO activity in immature chicks were best characterized by a segmented response function consisting of a parabola joined to a straight line. The maximum value was estimated to occur at 13 days and from day 16 declined linearly. The functional relationship did not provide evidence of an early lag period or a peak at 4 weeks in immature chickens. Food deprivation in matched groups of birds demonstrated the effect of food on GLO activity in chickens. Food and water deprivation for 12 h and food deprivation for 24 h had no significant effect on GLO activity. Food deprivation for 48 h caused a significant decrease in GLO activity and it was not further depressed by deprivation for 72 h. Initial GLO activity was restored within 24 h of repletion. The results clearly demonstrated that gender, age, and starvation are determinants of ascorbate synthesis in chickens.

Examination of the qualitative ability of some cold water marine teleosts to synthesise ascorbic acid

Activity of l-gulonolactone oxidase (GLO), which is involved in ascorbic acid (AA) synthesis, was measured in kidney and liver tissues of seven teleosts, Atlantic sterlet, Acipenser ruthenus, and Atlantic dogfish, Squalus acanthias. GLO activity was also measured in liver and kidney tissues of Atlantic halibut, Hippoglossus hippoglossus, and Atlantic salmon, Salmo salar, fed AA unsupplemented and AA supplemented diets. AA synthesis was not detected in both tissues of the seven teleosts but was found in the kidneys of sterlet and dogfish. The respective GLO activities were 240 and 513 μg AA synthesised g −1 kidney tissue h −1. Cold water marine teleosts such as herring, Clupea harengus, eel, Anguilla anguilla, salmon, cod, Gadus morhua, mackerel, Scomber scombrus, halibut, and turbot, Scophthalmus maximus, lack GLO activity and therefore dietary AA supplementation is required in their culture. AA deficient as well as AA supplemented diets did not induce GLO activity in salmon or halibut.

Expression of rat gene for l-gulono- γ-lactone oxidase, the key enzyme of l-ascorbic acid biosynthesis, in guinea pig cells and in teleost fish rainbow trout ( Oncorhynchus mykiss)

The ability of rainbow trout liver and kidney preparations to produce l-ascorbic acid with an added source of l-gulono- γ-lactone oxidase (GLO) and the absence of their own GLO activity suggested that the reason for the absence of l-ascorbic acid biosynthesis in fish and in guinea pig, a scurvy-prone mammal, can be similar. Nevertheless, results of rat GLO cDNA expression in guinea pig cells and in rainbow trout proved different. In guinea pig cells, rat GLO was expressed in a functional form. Regardless of recombinant GLO transcripts detected in rainbow trout embryos, alevins and in juvenile fish, neither GLO protein nor GLO activity were found. Furthermore, production of l-ascorbic acid in transgenic rainbow trout was not revealed in feeding tests with vitamin C-free diets or after direct administration of l-gulono- γ-lactone. These results indicate that conditions required for translation or stability of rat GLO are absent in rainbow trout tissues.

L-gulono-gamma-lactone oxidase is not induced in rats by xenobiotics stimulating L-ascorbic acid biosynthesis.

L-Ascorbic acid (AsA) is synthesized from D-glucose in rats; the terminal step of this synthetic pathway is catalyzed by L-gulono-gamma-lactone oxidase (GLO). In this study, we examined the effects of phenobarbital (PB) and 3-methylcholanthrene (MC), both of which are known to stimulate AsA biosynthesis in rats, on the hepatic levels of GLO activity, GLO mRNA, and AsA. Firstly, the existence of GLO mRNA was examined in the liver, kidney, lung, small intestine, spleen, testis, and prostate from a male rat; and GLO mRNA was found to be present only in the liver, in which GLO activity was also detected. The intraperitoneal injection with PB (100 mg/day/kg body weight, once a day for 2 days) or MC (20 mg/day/kg body weight, once) significantly elevated the hepatic level of AsA and the urinary excretion of AsA in rats (5-week-old males). The hepatic levels of cytochrome P-450IIB1 mRNA and cytochrome P-450IIB2 mRNA and those of cytochrome P-450IA1 mRNA and cytochrome P-450IA2 mRNA were also elevated in the rats treated with PB and MC, respectively, indicating a normal response of these animals to these compounds. However, the level of GLO mRNA and the activity of GLO in the liver tended to be slightly decreased by the administration of PB or MC, though the differences were not significant. Thus it is clear that the treatment with PB or MC stimulates the biosynthesis of AsA by increasing the activity of some enzyme(s) participating in the synthesis prior to GLO.

Expression in monkey cells of the missing enzyme in L-ascorbic acid biosynthesis, L-Gulono-γ-lactone oxidase

L-Gulono-γ-lactone oxidase (GLO), an enzyme that is missing in scurvy-prone animals, was produced in COS-1 cells by transfection with a minigene constructed from pSVL vector and rat GLO cDNA. A functional GLO protein was expressed, which was indistinguishable from rat GLO in molecular size. The microsomes obtained from the transfected COS-1 cells showed GLO activity comparable to that in rat liver microsomes.

Glyoxalase 1 (GLO) in the chicken: genetic variation and lack of linkage to the MHC.

Electrophoretic variation of glyoxalase 1 (GLO) has been detected in chicken red-cell lysates. Three phenotypes are shown to be inherited through a diallelic system, just as in humans and mice. The chicken GLO phenotype differ from their mammalian counterparts in that one of the homozygotes is devoid of GLO activity. The heterozygote produces two bands, while the other homozygote yields a single band of GLO activity with mobility equal to the faster of these two bands. In noninbred White Leghorn birds, the GLO2 allele occurred significantly more often in birds homozygous for the B1 allele at the chicken MHC than in those homozygous for B19, suggesting that the products of these loci may have population associations in the chicken. Absence of close linkage between the GLO and B loci was, however, demonstrated by appropriate test crosses.

EVOLUTION OF AN ENZYME SYSTEM: ASCORBIC ACID BIOSYNTHESIS IN MONOTREMES AND MARSUPIALS.

L-ascorbic acid, vitamin C for those species dependent on an exogenous source, is a physiologically essential molecule for all multicellular animals. Nevertheless, some species of birds and mammals lack and apparently have lost the capacity to synthesize it. Some vertebrate species synthesize it in liver, some in kidney, and some in both. It has been the goal of our research for several years to understand the evolution of the ascorbic acid pathway in vertebrates, as a model for the study of (1) regressive (=streamlining) evolution, (2) tissue locus shifts in biochemical pathways, and (3) the operation of selection on the ability of animals to regulate the production of an essential molecule. We here present data on the activity and tissue locus of L-gulonolactone oxidase (GLO) (EC1.1.3.8) in the Monotremata and Marsupialia and integrate these data into an hypothesis of the evolution of ascorbate biosynthesis in vertebrates. GLO catalyzes the final step, conversion of L-gulonolactone to L-ascorbic acid, in the pathway by which the latter is synthesized from D-glucose (Dagley and Nicholson, 1970; Chatterjee, 1973a). Although few species of most higher taxa have been examined, invertebrates (Gupta et al., 1972; Chatterjee, 1973b), teleost fish (Chatterjee, 1973a; Andrews and Murai, 1975), some birds (Chaudhuri and Chatterjee, 1969), and a few mammals (Chatterjee, 1973a), appear to lack GLO, and thus are unable to synthesize ascorbate. Lungfish (D. E. Dykhuizen, pers. comm.), amphibians, reptiles, and most birds are reported to have GLO in the kidney whereas eutherian mammals and some birds have it in the liver (Chatterjee, 1973a). We recently demonstrated (Birney et al., 1979) GLO activity in kidney of monotremes and in both liver and kidney in bandicoots and in some macropods. Prior to that report, the Virginia opossum (Didelphis virginiana) was the only noneutherian mammal studied, and it was believed to be capable of synthesizing ascorbate in liver on the basis of dehydrogenation of L-gulonolactone as determined histologically (Nakajima et al., 1969). The basic evolutionary relationship and times of divergence for the three major groups of mammals, Prototheria, Metatheria, and Eutheria, are reasonably well understood (Hopson and Crompton, 1969; Crompton and Jenkins, 1973; Lillegraven, 1974; Clemens, 1977). Furthermore, Ride (1964), Kirsch (1977a, 1977b), and Kirsch and Calaby (1977) have greatly enhanced our knowledge of the evolutionary relationships within the Marsupialia. Thus we feel that the relationships of the major groups of mammals discussed herein are adequately known so that we can superimpose the pattern of GLO activity on this phylogeny and initiate an understanding of the evolution of the biochemical system involved by proposing and evaluating some preliminary hypotheses.

Evidence for a 'silent allele' GLO0 at the glyoxalase I locus.

In a three-generation family, the segregation of an apparent silent allele at the GLO I locus in association with the rare HLA haplotype 'AW30-CW4-BW35' was observed in four members. In two cases the assumption of homozygosity at the GLO locus would lead to mother-child exclusions. Phenotypically, the GLO activity in the GLO0 carriers is clearly diminished.

National Parks in Quebec

Activity of l-gulonolactone oxidase (GLO), which is involved in ascorbic acid (AA) synthesis, was measured in kidney and liver tissues of seven teleosts, Atlantic sterlet, Acipenser ruthenus, and Atlantic dogfish, Squalus acanthias. GLO activity was also measured in liver and kidney tissues of Atlantic halibut, Hippoglossus hippoglossus, and Atlantic salmon, Salmo salar, fed AA unsupplemented and AA supplemented diets. AA synthesis was not detected in both tissues of the seven teleosts but was found in the kidneys of sterlet and dogfish. The respective GLO activities were 240 and 513 μg AA synthesised g−1 kidney tissue h−1. Cold water marine teleosts such as herring, Clupea harengus, eel, Anguilla anguilla, salmon, cod, Gadus morhua, mackerel, Scomber scombrus, halibut, and turbot, Scophthalmus maximus, lack GLO activity and therefore dietary AA supplementation is required in their culture. AA deficient as well as AA supplemented diets did not induce GLO activity in salmon or halibut.