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PubMed2002

In situ staining of activities of enzymes involved in carbohydrate metabolism in plant tissues.

A powerful technique is described to localize the activities of a range of enzymes in a wide variety of plant tissues. The method is based on the coupling of the enzymatic reaction to the reduction of NAD and subsequent reduction and precipitation of nitroblue tetrazolium. Enzymes that did not reduce NAD could be visualized by coupling their activities to glucose-6-phosphate dehydrogenase activity via one or more intermediary 'coupling' enzymes. The method is shown to be applicable for the detection of the activities of hexokinase, fructokinase, sucrose synthase, uridine 5'-diphospho-glucose pyrophosphorylase, ADP-glucose pyrophosphorylase, phosphoglucomutase, and phosphoglucose isomerase. It could be used for all tissues tested, including green leaves, stems, roots, fruits, and seeds. The method is specific, very sensitive, and has a high spatial resolution, giving information at the cellular and the subcellular level. The localization of sucrose synthase, invertase, and uridine 5'-diphospho-glucose pyrophosphorylase in transgenic potato plants, carrying a cytokinin biosynthesis gene, is studied and compared with wild-type plants.

Alkyl and Aryl Transferases↗
PubMed1990

Enzyme electrophoresis as an alternative method for separating the sympatric Leishmania vectors Phlebotomus papatasi and Phlebotomus langeroni (Diptera: Psychodidae).

Trials were conducted to determine the accuracy of separating the sympatric sand fly species Phlebotomus papatasi (Scopoli) and P. langeroni Nitzulescu by means of cellulose acetate enzyme electrophoresis. Malic enzyme, phosphoglucomutase, 6-phosphogluconate dehydrogenase, and fumarate hydratase were each evaluated in laboratory-reared and field-collected populations of the two species. Each of the four enzyme-based identifications was highly sensitive (greater than 97%) and specific (greater than 93%). Identifications based upon fumarate hydratase were in perfect agreement with morphological identifications, and evidence was obtained which indicates that this enzyme may be the most stable of the four enzymes tested. The application of enzyme-based vector identification is discussed in relation to classical and novel survey procedures for Leishmania promastigote detection in sand flies.

Animals↗
PubMed1976

Human neoplastic and normal cells in tissue culture. I. Cell lines derived from malignant melanomas and normal melanocytes.

Forty-six cell lines derived from 31 human melanomas obtained from 28 patients were cultured. Fourteen of 16 lines have produced malignant tumors when injected into nude (thymus-deficient) mice. Tumors in 5 of the nude mice metastasized to distant lymph nodes and/or to the lungs of the mouse host. Extreme variability from line to line was observed for doubling time (34 to 106 hr), plating efficiency (0-86%), and melanin production. All tested lines had type B glucose-6-phosphate dehydrogenase, thereby excluding HeLa cell contamination. HeLa cells have been grown for some time in our laboratory. Our results clearly demonstrated that HeLa cell contamination does not occur invariably in heteroploid lines growing in a laboratory simultaneously with Hela cells, provided that proper care is taken to avoid such occurrence. Multiple cell lines derived from the same tumor had identical phosphoglucomutase enzyme phenotype, which suggested a lack of significant cross-contamination between the lines. Four long-term cultures of normal human uveal embryo melanocytes have also been established and characterized. Although all produced melanin after reaching saturation density, they differed from the melanoma cells morphologically; they were flat, not refringent, and lacked piling up and plating ability. When melanoma cells were exposed to bromodeoxyuridine (BUDR) for long periods, a phenotypic change toward non-neoplastic characteristics was observed. Cells became flat and not refringent and, when injected into nude mice, tumors appeared after a long latent period. These changes were completely reversible in vitro and in vivo. The BUDR-treated cultures were undistinguishable from the untreated mother cultures after 2 to 3 passages. Lines derived from tumors in nude mice (obtained by injection of BUDR-treated cells) were again indistinguishable from the untreated mother line. Normal melanocytes were mostly euploid; all the melanoma cells were aneuploid. All 29 cell lines derived from 14 patients had an average chromosome number higher than 46. Detailed group-by-group chromosome analysis always showed an excess of C chromosomes, which suggested that hyperreduplication of one or more C chromosomes is a specific characteristic of human melanomas.

Adult↗
PubMedDate not supplied

Esterases in the malaria vector mosquito, Anopheles culicifacies. Genetic and linkage analyses of an alpha and beta polymorphism.

The genetic and linkage analyses of an alpha and beta esterase polymorphism in Anopheles culicifacies are presented. A survey of laboratory strains uncovered four electrophoretic variants for the alpha esterase and two for the beta esterase. Genetic analyses indicated that the variants of the alpha esterase are under the control of codominant alleles of a single locus and that this locus is linked to the locus controlling the expression of the codominant alleles of the beta esterases. The esterases are not linked either to sex (chromosome 1) or to maroon eye (chromosome 2) but to the chromosome 3 markers, dieldrin resistance and phosphoglucomutase. The gene sequence is Est-alpha--Dl--Pgm--Est-beta dnd the recombination frequencies are as follows: Est-alpha--Dl = 2.9 percent; Dl--Pgm = 5.8 percent; Pgm--Est-beta = 6.4 percent; Est-alpha--Est-beta = 15.1 percent, Est-alpha--Pgm = 8.7 percent and Dl--Est-beta = 12.2 percent.

Animals↗
PubMedDate not supplied

Linkage relationships among four enzyme loci in the predatory, treehole mosquito.

Estimates are reported for the linkage distances between the isozyme loci for hydroxybutyrate dehydrogenase (HbDH), phosphoglucomutase (PGM), phosphoglucose isomerase (PGI), and esterase 8 (Est 8) in the predatory, treehole mosquito, Toxorhynchites rutilus rutilus. Measured linkage distances suggest that these four loci form a single autosomal linkage group approximately 47.4 linkage units in length. The estimates were measured with error because of small family sizes and, perhaps, because of different rates of recombination between males and females or the presence of a paracentric inversion associated with this linkage group. The four loci are tentatively placed in the following order: Est 8--HbDH--PGM--PGI.

Animals↗
PubMed1984

Major morphological effects of a regulatory gene: Pgm1-t in rainbow trout.

We have investigated the morphological effects of a genetic locus, Pgm1-t, that affects the expression of a phosphoglucomutase locus (Pgm1) in liver of rainbow trout (Salmo gairdneri). We have previously shown that embryos with liver Pgm1 expression hatch earlier than those without liver Pgm1 expression. We predicted that this difference in developmental rate should cause a reduction in meristic counts in the more rapidly developing fish with liver Pgm1 expression. Eight meristic (countable) characters in nine full-sib groups segregating for the presence or absence of liver Pgm1 expression are in agreement with this prediction. In eight of the nine families, there is a significant difference in the multivariate distribution of the eight meristic counts between full sibs with and without liver Pgm1 expression. This separation in multivariate space is based on a tendency for lower meristic counts in fish with liver Pgm1 expression. The magnitude of these morphological differences is similar to that between two subspecies of cutthroat trout (Salmo clarki) that show substantial genetic divergence at structural loci encoding enzymes (Nei's D = 0.34). These data support the view that small changes in the developmental process caused by genetic differences at regulatory genes can have large effects on morphology.

Animals↗
PubMed2001

Reappraisal of the currently prevailing model of starch biosynthesis in photosynthetic tissues: a proposal involving the cytosolic production of ADP-glucose by sucrose synthase and occurrence of cyclic turnover of starch in the chloroplast.

A vast amount of information has accumulated which supports the view that sucrose and starch are end-products of two segregated, yet highly interconnected, gluconeogenic pathways taking place in the cytosol and chloroplast, respectively. However, several lines of experimental evidences indicate that, essentially identical to the case of heterotrophic tissues, starch formation in the photosynthetic tissues may involve the direct import to the chloroplast of cytosolic hexose (C6) units derived from the sucrose breakdown. This evidence is consistent with the idea that synthesis of a sizable pool of ADP-glucose takes place in the cytosol by means of sucrose synthase whereas, basically in agreement with recent investigations dealing with glycogen biosynthesis in bacteria and animals, chloroplastic phosphoglucomutase and ADP-glucose pyrophosphorylase are most likely playing a role in channelling of glucose units derived from the starch breakdown in the chloroplast, thus making up a regulatory starch turnover cycle. According to this new view, we propose that starch production in the chloroplast is the result of a flexible and dynamic mechanism wherein both catabolic and anabolic reactions take place simultaneously in a highly interactive manner. Starch is seen as an intermediate component of a cyclic gluconeogenic pathway which, in turn, is connected with other metabolic pathways. The possible importance of metabolic turnover as a way to control starch production is exemplified with the recently discovered ADP-glucose pyrophosphatase, an enzyme likely having a dual role in controlling levels of ADP-glucose linked to starch biosynthesis and diverting carbon flow towards other metabolic pathways.

Adenosine Diphosphate Glucose↗
PubMed2005

Gravitropism in leaves of Arabidopsis thaliana (L.) Heynh.

In higher plants, stems and roots show negative and positive gravitropism, respectively. However, current knowledge on the graviresponse of leaves is lacking. In this study, we analyzed the positioning and movement of rosette leaves of Arabidopsis thaliana under light and dark conditions. We found that the radial positioning of rosette leaves was not affected by the direction of gravity under continuous white light. In contrast, when plants were shifted to darkness, the leaves moved upwards, suggesting negative gravitropism. Analysis of the phosphoglucomutase and shoot gravitropism 2-1 mutants revealed that the sedimenting amyloplasts in the leaf petiole are important for gravity perception, as is the case in stems and roots. In addition, our detailed physiological analyses revealed a unique feature of leaf movement after the shift to darkness, i.e. movement could be divided into negative gravitropism and nastic movement. The orientation of rosette leaves is ascribed to a combination of these movements.

Arabidopsis↗
PubMed2003

Glucose-6-phosphate isomerase is not a specific autoantigen in rheumatoid arthritis.

OBJECTIVE: To test the hypothesis that glucose-6-phosphate isomerase (GPI) is a novel autoantigen in RA. METHODS: Eighty-eight serum samples from 23 patients with rheumatoid arthritis (RA), 25 with Sjögren's syndrome, 20 with systemic lupus erythematosus and 20 healthy controls were tested by enzyme-linked immunosorbent assay (ELISA) using a commercially available, partially purified rabbit GPI as antigen. Beside each duplicate well containing antigen (10 micro g/ml), uncoated blocked duplicate wells (phosphate-buffered saline only) were included as controls for non-specific binding for every serum tested. We also examined antibodies binding to various polypeptides in the GPI preparation by immunoblotting in 73 of the sera. RESULTS: By ELISA, binding levels were low and there was no difference between serum from patients with RA, other rheumatic diseases and normal controls. By immunoblotting, antibodies binding to the GPI polypeptide were present in 70-80% of all groups tested. In addition, we showed that another polypeptide identified as phosphoglucomutase was also present in the preparation and reacted with human immunoglobulins. CONCLUSION: Our findings suggest that GPI is not a specific autoantigen in RA.

Antibodies↗
PubMed2004

Thermal selection of PGM allozymes in newly founded populations of the thermotolerant vent polychaete Alvinella pompejana.

Alvinella pompejana lives on the top of chimneys at deep-sea hydrothermal vents of the East Pacific Rise. It is thought to be one of the most thermotolerant and eurythermal metazoans. Our experimental approach combines methods of population genetics and biochemistry, considering temperature as a potential selective factor. Phosphoglucomutase (Pgm-1 locus) is one of the most polymorphic loci of A. pompejana and exhibits four alleles, from which alleles 90 and 100 dominate with frequencies of approximately 0.5 in populations. Results from previous studies suggested that allele 90 might be more thermostable than allele 100. Significant genetic differentiation was found by comparing contrasted microhabitats, especially the young, still hot, versus older and colder chimneys, with allele 90 being at highest frequency on young chimneys. Moreover the frequency of allele 90 was positively correlated with mean temperature at the opening of Alvinella tubes. In parallel, thermostability and thermal optimum experiments demonstrated that allele 90 is more thermostable and more active at higher temperatures than allele 100. This dataset supports an additive model of diversifying selection in which allele 90 is favoured on young hot chimneys but counterbalanced over the whole metapopulation by the dynamics of the vent ecosystem.

Animals↗
PubMed1978

Biochemical study of certain enzymes and metabolites of the carbohydrate metabolism in the skeletal muscle of the dengue virus-infected mice.

Changes in enzymes and metabolites of the carbohydrate metabolism in skeletal muscles were studied in mice after intracerebral inoculation of dengue type 2 virus. It was noted that lactic dehydrogenase, aldolase, phosphoglucoisomerase, phosphoglucomutase, GO-T and GP-T activity were enhanced initially by two- to three-fold, reaching a peak on day 5. As the illness appeared in mice, all the enzyme activities were lowered and were about three times less in the paralytic stage on the 8th day as compared to controls. Fructose-1,6-diphosphatase activity was increased on the 4th and 5th days but decreased later. Acid phosphatase increased abruptly from the 6th day while alkaline phosphatase activity was irregular. Creatine increased on the 4th and 5th days but diminished later. Glycogen decreased from the beginning and was lowest on the 5th day, but the levels increased later and were maximum in paralysed muscles. On the other hand, lactic acid began accumulating in the muscles and was maximum on the 5th day, then declined. Dengue virus was detected in the muscles from the 2nd day but higher titres were seen from the 6th day. Changes similar to the preparalytic stage of mice may occur in human beings, causing myalgia.

Acid Phosphatase↗
PubMed1987

The gtaB marker in Bacillus subtilis 168 is associated with a deficiency in UDPglucose pyrophosphorylase.

Fifty-six mutants of Bacillus subtilis 168 were selected for resistance to bacteriophages phi 29 or phi 25. The mutations were all linked to previously described teichoic acid markers gtaA, gtaB or gtaC, for the first and last of which, the gene products have previously been identified. Each linkage group was shown to have two distinct phenotypes with respect to phage resistance and cell-wall galactosamine content. Recombination indexes of 0.35, 0.13 and 0.41 for groups A, B and C respectively were consistent with the presence of two average-sized genes in groups A and C. Correlation between genetic and phenotypic differences supported this conclusion and led to the designation of two new markers, gtaD and gtaE. Two- and three-factor transformation crosses suggested the order hisA-gtaB-gtaD-gtaA-tag-1 and gtaC-gtaE-argC. Assays for UDPglucose pyrophosphorylase and phosphoglucomutase activities in soluble extracts of representative mutants revealed that, in contrast to previous findings, the former activity was virtually undetectable in all nine group B mutants examined, suggesting that gtaB is the structural gene of this enzyme. Our results allow us to account for discrepancies with respect to previous reports. The thermosensitive mutation previously designated rodC1 was shown to be 90% cotransformable with tag-1. In view of their extremely similar phenotypes the former mutation was renamed tag-3, and the likely order obtained was gtaA-tag-3-tag-1. This suggests that many mutations associated with deformation of cell shape in B. subtilis are located in the region where teichoic acid genes map.

Bacillus subtilis↗
PubMed1976

The uptake and metabolism of glucose, maltose and starch by the rumen ciliate Epidinium ecaudatum caudatum.

[14C]Glucose taken up by Epidinium ecaudatum caudatum was found in the pool, in the protozoal polysaccharide and in the bacteria associated with the protozoa. The amount incorporated into the polysaccharide depended on the square of the glucose concentration. Evidence was obtained that glucose was probably taken up initially into the pool unchanged, and then rapidly converted into glucose 6-phosphate and maltose which were subsequently hydrolysed to glucose. [14C]-Maltose was taken up at 20 to 30% of the rate of [14C]glucose, with 14C appearing initially in maltose and glucose 6-phosphate. 14C from 14C-labelled soluble starch appeared in the pool as maltose, glucose 6-phosphate and glucose in that order, but incorporation into protozoal polysaccaride was poor. Hexokinase, phosphoglucomutase, alpha-glucan and maltose phosphorylases, glucose 6-phosphatase and maltase activities were found in the protozoa.

Animals↗
PubMed2003

The ARG1-LIKE2 gene of Arabidopsis functions in a gravity signal transduction pathway that is genetically distinct from the PGM pathway.

The arl2 mutants of Arabidopsis display altered root and hypocotyl gravitropism, whereas their inflorescence stems are fully gravitropic. Interestingly, mutant roots respond like the wild type to phytohormones and an inhibitor of polar auxin transport. Also, their cap columella cells accumulate starch similarly to wild-type cells, and mutant hypocotyls display strong phototropic responses to lateral light stimulation. The ARL2 gene encodes a DnaJ-like protein similar to ARG1, another protein previously implicated in gravity signal transduction in Arabidopsis seedlings. ARL2 is expressed at low levels in all organs of seedlings and plants. arl2-1 arg1-2 double mutant roots display kinetics of gravitropism similar to those of single mutants. However, double mutants carrying both arl2-1 and pgm-1 (a mutation in the starch-biosynthetic gene PHOSPHOGLUCOMUTASE) at the homozygous state display a more pronounced root gravitropic defect than the single mutants. On the other hand, seedlings with a null mutation in ARL1, a paralog of ARG1 and ARL2, behave similarly to the wild type in gravitropism and other related assays. Taken together, the results suggest that ARG1 and ARL2 function in the same gravity signal transduction pathway in the hypocotyl and root of Arabidopsis seedlings, distinct from the pathway involving PGM.

Amino Acid Sequence↗
PubMed2003

Histochemical analysis reveals organ-specific quantitative trait loci for enzyme activities in Arabidopsis.

To identify genetic loci involved in the regulation of organ-specific enzyme activities, a specific histochemical staining protocol was used in combination with quantitative trait locus (QTL) analysis. Using phosphoglucomutase (PGM) as an example, it is shown that enzyme activity can specifically, and with high resolution, be visualized in non-sectioned seedlings of Arabidopsis. The intensities of staining were converted to quantitative data and used as trait for QTL analysis using Landsberg erecta x Cape Verde Islands recombinant inbred lines. Independently, PGM activities were quantified in whole-seedling extracts, and these data were also used for QTL analysis. On the basis of extract data, six significant (P < 0.05) loci affecting PGM activity were found. From the histochemical data, one or more specific QTLs were found for each organ analyzed (cotyledons, shoot apex, hypocotyl, root, root neck, root tip, and root hairs). Loci detected for PGM activity in extracts colocated with loci for histochemical staining. QTLs were found coinciding with positions of (putative) PGM genes but also at other positions, the latter ones supposedly pointing toward regulatory genes. Some of this type of loci were also organ specific. It is concluded that QTL analysis based on histochemical data is feasible and may reveal organ-specific loci involved in the regulation of metabolic pathways.

Alleles↗
PubMed2005

Sucrose-specific induction of the anthocyanin biosynthetic pathway in Arabidopsis.

Sugars act as signaling molecules, whose signal transduction pathways may lead to the activation or inactivation of gene expression. Whole-genome transcript profiling reveals that the flavonoid and anthocyanin biosynthetic pathways are strongly up-regulated following sucrose (Suc) treatment. Besides mRNA accumulation, Suc affects both flavonoid and anthocyanin contents. We investigated the effects of sugars (Suc, glucose, and fructose) on genes coding for flavonoid and anthocyanin biosynthetic enzymes in Arabidopsis (Arabidopsis thaliana). The results indicate that the sugar-dependent up-regulation of the anthocyanin synthesis pathway is Suc specific. An altered induction of several anthocyanin biosynthetic genes, consistent with in vivo sugar modulation of mRNA accumulation, is observed in the phosphoglucomutase Arabidopsis mutant accumulating high levels of soluble sugars.

Anthocyanins↗
PubMed2000

Cryptic female choice in the yellow dung fly Scathophaga stercoraria (L.).

Both female choice and male-male competition may take place during reproduction in many species. Female choice tends to be less obvious than male-male competition and consequently has received less attention from researchers. The opportunity for cryptic female choice arises after multiple insemination. Through postcopulatory processes, a female could alter the pattern of paternity among her offspring so that it does not directly reflect the different contributions of sperm made by her mates. To be able to determine if a female alters the relative sperm contributions of her mates, the behaviors and influences of the males must therefore be first taken into account. The interest of each male is to father all the offspring, and the interest of each female is to maximize paternal quality. Female yellow dung flies have complex internal reproductive tracts that may give them considerable control over the fertilization success of stored sperm from different males. In laboratory trials to date, the last male to mate has usually been most successful. In the present study, cryptic choice occurred in Scathophaga stercoraria and the pattern of choice was consistent with previously reported results. The fertilization success of a female's second mate (P2) was substantially larger if a female was kept at constant temperature and if the second male was genetically similar to her at the phosphoglucomutase (Pgm) locus. Females from the field normally have three spermathecae, but some have four. Lines were bred to have either three or four spermathecae. Flies from the different lines were crossed to generate females with similar genetic backgrounds that had either three or four spermathecae. P2 was significantly lower for high-quality females, that is, those that laid larger-than-average-clutches, with four spermathecae than for low-quality females with four spermathecae; female quality had no influence on P2 for females with three spermathecae. The results suggest that only large females may benefit from increased spermathecae number by being able to act against male interests. Females may only have three spermathecae, even though genetic variation for more is present, because selection for more spermathecae is weak.

Animals↗
PubMed2002

Allele frequency shifts in response to climate change and physiological consequences of allozyme variation in a montane insect.

Rapid changes in climate may impose strong selective pressures on organisms. Evolutionary responses to climate change have been observed in natural populations, yet no example has been documented for a metabolic enzyme locus. Furthermore, few studies have linked physiological responses to stress with allozyme genotypic variation. We quantified changes in allele frequency between 1988 and 1996 at three allozyme loci (isocitrate dehydrogenase, Idh; phosphoglucose isomerase, Pgi; and phosphoglucomutase, Pgm) for the leaf beetle Chrysomela aeneicollis in the Bishop Creek region of the Sierra Nevada of California (2900-3300 m). Beetles often experience high daytime (> 32 degrees C) and extremely low nighttime (< -5 degrees C) temperatures during summer. Bishop Creek weather station data indicated that conditions were unusually dry before 1988, and that conditions were cool and wet during the years preceding the 1996 collection. We found directional changes in allele frequency at Pgi (11% increase in the Pgi-1 allele), but not at Idh or Pgm. We also found that physiological response to thermal extremes depended on Pgi genotype. Pgi 1-1 individuals induced expression of a 70-kD heat shock protein (HSP) at lower temperatures than 1-4 or 4-4 individuals, and 1-1 individuals expressed higher levels of HSP70 after laboratory exposure to temperatures routinely experienced in nature. Survival after nighttime laboratory exposure to subzero temperatures depended on gender, previous exposure to cold, and Pgi genotype. Females expressed higher levels of HSP70 than males after exposure to heat, and recovery by female Pgi 1-1 homozygotes after exposure to cold (-5 degrees C) was significantly better than 1-4 or 4-4 genotypes. These data suggest that the cooler climate of the mid-1990s may have caused an increase in frequency of the Pgi-1 allele, due to a more robust physiological response to cold by Pgi 1-1 and 1-4 genotypes.

Animals↗