Canine erythrocyte pyruvate kinase. II. Properties of the abnormal enzyme associated with hemolytic anemia in the basenji dog

We have compared the solubility, kinetic, immunological, and electrophoretic properties of erythrocyte pyruvate kinase from normal dogs and Basenji dogs with congenital hemolytic anemia due to pyruvate kinase deficiency. Differences can be detected between the two enzymes by all methods. The enzyme from the affected animals has a greater solubility in ammonium sulfate. It has a lower K _m for phosphoenolpyruvate, while the K _m for ADP is increased. This enzyme is not inhibited by ATP or activated by fructose 1,6-diphosphate. The enzyme from the affected animals has none of the allosteric properties characteristic of the normal canine enzyme. No difference can be detected by enzyme inactivation with rabbit antiserum against the human erythrocyte enzyme, but a slight spur is observed on comparison of the two enzymes by Ouchterlony immunodiffusion. The enzymes also differ in their electrophoretic mobilities on starch gel electrophoresis.     

Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes

Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi‐copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxonomic specificity and sensitivity of qPCR assays targeting the rodA gene (rodA984) and two regions of the multi‐copy 23S ribosomal RNA gene (EC23S and EC23S857). Experimental analyses of 28 culture collection strains representing E. coli and 21 related non‐target species indicated that the uidA405 and rodA984 assays were both 100% specific for E. coli while the EC23S assay was only 29% specific. The EC23S857 assay was only 95% specific due to detection of E. fergusonii. The uidA405, rodA984, EC23S and EC23S857 assays were 85%, 85%, 100% and 86% sensitive, respectively, in detecting 175 presumptive E. coli culture isolates from fresh, marine and waste water samples. In analyses of DNA extracts from 32 fresh, marine and waste water samples, the rodA984, EC23S and EC23S857 assays detected mean densities of target sequences at ratios of approximately 1 : 1, 243 : 1 and 6 : 1 compared with the mean densities detected by the uidA405 assay. Conclusions: The EC23S assay was less specific for E. coli, whereas the rodA984 and EC23S857 assay taxonomic specificities and sensitivities were similar to those of the uidA405 gene assay. Significance and Impact: The EC23S857 assay has a lower limit of detection for E. coli cells than the uidA405 and rodA984 assays due to its multi‐copy gene target and therefore provides greater analytical sensitivity in monitoring for these faecal pollution indicators in environmental waters by qPCR methods.     

Effect of temperature on seed production in the invasive grass Glyceria maxima (Hartm.) Holmb. (Poaceae) in South Africa

Temperature is one of the main factors that determine sexual reproduction in terrestrial and emergent aquatic plant species. The effect of temperature on sexual reproduction and seed production of Glyceria maxima (Hartm.) Holmb. in the southern hemisphere is unknown. Glyceria maxima collections in February 2010 at three isolated infestations in KwaZulu-Natal failed to yield a single seed, only empty panicles. Laboratory experiments showed that vernalisation had no consistent effect on seed production. Field- and laboratory-grown plants produced seeds in the 2010/2011 season, because of having sufficient time at optimum temperatures required for seed production (1 491 and 1 585 hours, respectively), compared to a shorter period (1 352 hours) of suitable temperatures during the 2009/2010 growing season. An inadequate period of optimum temperatures (15–25°C) during seed production resulted in the lack of seeds in the field in the 2009/2010 growing season. This study showed that temperature and duration of exposure thereto during the seed-production period play vital roles in G. maxima sexual reproduction.     

3q26.31–q29 duplication and 9q34.3 microdeletion associated with omphalocele,ventricular septal defect,abnormal first-trimester maternal serum screening and increased nuchal translucency: Prenatal diagnosis and aCGH characterization

We present prenatal diagnosis and array comparative genomic hybridization characterization of 3q26.31–q29 duplication and 9q34.3 microdeletion in a fetus with omphalocele, ventricular septal defect, increased nuchal translucency, abnormal first-trimester maternal screening and facial dysmorphism with distinct features of the 3q duplication syndrome and Kleefstra syndrome. The 26.61-Mb duplication of 3q26.31–q29 encompasses EPHB3, CLDN1 and CLDN16, and the 972-kb deletion of 9q34.3 encompasses EHMT1. We review the literature of partial trisomy 3q associated with omphalocele and discuss the genotype–phenotype correlation in this case.     

Constituents from the Formosan apple reduce tyrosinase activity in human epidermal melanocytes

Tyrosinase is a copper-containing monooxygenase that catalyzes melanin synthesis in skin melanocytes. Herein, 13 compounds from the Formosan apple (Malus doumeri var. formosana), an indigenous Taiwanese plant, were isolated and identified. The active constituents were identified as 3-hydroxyphloretin (7) and catechol (9); they exhibited potent hydroxyl radical-scavenging (IC₅₀ values, 0.6 and 1.1 μM) and cellular tyrosinase-reducing activities (IC₅₀ values, 32 and 22 μM) in human epidermal melanocytes. In addition, we evaluated the level of several tyrosinase-related proteins by Western blot analysis. In contrast to 3-hydroxyphloretin (7), which showed no effect on the level of these proteins, catechol (9) reduced their activity and the expression of the respective genes, as determined by quantitative real-time PCR. In a kinetic analysis of mushroom tyrosinase, 3-hydroxyphloretin (7) was a competitive inhibitor. These two constituents exhibited metal-coordinating interactions with copper ions in a virtual model of molecular docking with human tyrosinase. Thus, 3-hydroxyphloretin (7) and catechol (9) were the most active constituents from the Formosan apple; they exhibited anti-oxidant and tyrosinase reducing activities, suggesting their possible use as cosmetic agents.     

Pure interstitial duplication of chromosome 7q (7q31.2-->q33) in a 4-year-old girl with growth restriction, short stature, speech delay and intellectual disability

We report the cytogenetic and molecular characterization of a 22.3-Mb pure interstitial duplication of chromosome 7q, dup(7)(q31.2-->q33) in a 4-year-old girl with growth restriction, short stature, speech delay, inguinal hernia, strabismus and intellectual disability. We speculate that the gene dosage increase effect of the ING3 and LEP genes may be partially responsible for the phenotype of growth restriction and short stature in this patient.     

A comparison of maternal age, sex ratio and associated anomalies among numerically aneuploid, structurally aneuploid and euploid holoprosencephaly

During the period January 1987-July 2003, 59 cases of perinatally detected holoprosencephaly (HPE) with cytogenetic results were identified among 97,306 deliveries at Mackay Memorial Hospital. Among these 59 cases with HPE, 25 had euploidy, 27 had numerical aneuploidy, and 7 had structural aneuploidy. In the euploid cases, the male:female sex ratio was 0.39:1, whereas in the aneuploid cases, the ratio was 1:1. The mean (+/-SD) maternal ages for numerical aneuploidy, structural aneuploidy, and euploidy were 33.0 +/- 5.1 years, 27.9 +/- 2.1 years, and 27.8 +/- 5.0 years, respectively. The frequencies of associated major structural anomalies other than craniofacial defects in the cases with numerical aneuploidy, structural aneuploidy, and euploidy were 85.7%, 0%, and 16%, respectively. The present study of HPE suggests that a female excess appears only in the euploid cases, and advanced maternal age and structural anomalies are more commonly associated with the numerically aneuploid cases than the structurally aneuploid and euploid cases.     

Synthesis and antipicornavirus activity of (R)- and (S)-1-[5-(4'-chlorobiphenyl-4-yloxy)-3-methylpentyl]-3-pyridin-4-yl-imidazolidin-2-one

The new pyridyl imidazolidinone derivative, 1-[5-(4'-chlorobiphenyl-4-yloxy)-3-methylpentyl]-3-pyridin-4-yl-imidazolidin-2-one (+/-)-1a, was synthesized and found to have an excellent antiviral activity against EV71 (IC50 = 0.009 microM). Therefore, both the enantiomers, (S)-(+)-1a and (R)-(-)-1a, have been prepared starting from readily available monomethyl (R)-3-methylglutarate (7) as a useful chiral building block and their antiviral activity was evaluated in a plaque reduction assay. Interestingly, we observed that the enantiomer (S)-(+)-1a was 10-fold more active against enterovirus71 (EV71) (IC50 = 0.003 microM) than the corresponding enantiomer (R)-(-)-1a (IC50 = 0.033 microM). Similar results were found against all five strains (1743, 2086, 2231, 4643, and BrCr) of EV71 tested. This demonstrated that the absolute configuration of the chiral carbon atom at the 3-position of the alkyl linker considerably influenced the anti-EV71 activity of these pyridyl imidazolidinones.