Allele-specific Characterization of Alanine: Glyoxylate Aminotransferase Variants Associated with Primary Hyperoxaluria

Primary Hyperoxaluria Type 1 (PH1) is a rare autosomal recessive kidney stone disease caused by deficiency of the peroxisomal enzyme alanine: glyoxylate aminotransferase (AGT), which is involved in glyoxylate detoxification. Over 75 different missense mutations in AGT have been found associated with PH1. While some of the mutations have been found to affect enzyme activity, stability, and/or localization, approximately half of these mutations are completely uncharacterized. In this study, we sought to systematically characterize AGT missense mutations associated with PH1. To facilitate analysis, we used two high-throughput yeast-based assays: one that assesses AGT specific activity, and one that assesses protein stability. Approximately 30% of PH1-associated missense mutations are found in conjunction with a minor allele polymorphic variant, which can interact to elicit complex effects on protein stability and trafficking. To better understand this allele interaction, we functionally characterized each of 34 mutants on both the major (wild-type) and minor allele backgrounds, identifying mutations that synergize with the minor allele. We classify these mutants into four distinct categories depending on activity/stability results in the different alleles. Twelve mutants were found to display reduced activity in combination with the minor allele, compared with the major allele background. When mapped on the AGT dimer structure, these mutants reveal localized regions of the protein that appear particularly sensitive to interactions with the minor allele variant. While the majority of the deleterious effects on activity in the minor allele can be attributed to synergistic interaction affecting protein stability, we identify one mutation, E274D, that appears to specifically affect activity when in combination with the minor allele.     

Zinc Deficiency and Anther Development in Maize

With the onset of male reproductive phase at 28 days, zinc waswithdrawn from fifty percent of maize (Zea mays L. cv. Ganga2) plants grown in refined sand at 0.13 mg Zn liter^–1.Plants from which zinc was withdrawn developed zinc deficiencysymptoms in young leaves after 38 days and were low in tissuezinc. Their tassel formation and pollen development was retarded.Anthers failed to develop beyond freshly liberated young pollengrain stage and vessels were formed in place of sporogenoustissue in sixty percent anthers of the younger of the two florets.Anthers from these plants showed low zinc concentration andstimulated specific activities of catalase, peroxidase, ribonucleaseand acid phosphatase. On resuming normal zinc (0.13 mg Zn liter^–1) through rootsto the plants from which it was withdrawn for 17 days, vegetativegrowth was partially renewed and short axillary buds were formedbut the development of anthers remained retarded. (Received April 11, 1986; Accepted October 15, 1986)     

Mercury-Induced Inhibition of Photosystem II Activity and Changes in the Emission of Fluorescence from Phycobilisomes in Intact Cells of the Cyanobacterium, Spirulina platensis

Addition of low concentrations of mercury chloride (HgCl₂ tointact cells of the cyanobacterium, Spirulina platensis causedan enhancement in the intensity of fluorescence emitted fromphycocyanin at room temperature and induced blue shifts in theemission peak suggestive of changes in energy transfer withinthe phycobilisomes. HgCl₂ also suppressed the whole-chain electrontransport activity (H₂O methylviologen) at much lower concentrationsthan that required to inhibit Hill activity supported by para-benzoquinone.The extent of inhibition of Hill activity was much higher underhigh-intensity light than that under low-intensity light. Ourresults indicate that mercury ions at low concentrations affectthe transfer of energy within phycobilisomes and at high concentrationsthey inhibit electron transport in this cyanobacterium. (Received February 21, 1989; Accepted October 2, 1989)     

Bioenergetic consequences of protein overexpression in Saccharomyces cerevisiae

Summary Some bioenergetic consequences of overexpression of plasmid-encoded homologous (phosphoglycerate kinase), and heterologous (prochymosin), protein in S. cerevisiae strains grown in chemostat culture have been investigated. Both overexpressing strains were found to exhibit similar fermentation patterns despite a 10-fold difference in product expression levels. Biomass yields were lower than those for a control strain, and the onset of oxido-fermentative metabolism occurred at a lower dilution rate. A marked rise in cellular ATP content with increasing dilution rate during oxidative growth was observed in the strain overexpressing yeast phosphoglycerate kinase (PGK); this at present cannot be adequately explained. The inorganic phosphate content of the overexpressing strains was higher than that of the control and the phosphorylation potential of the prochymosin expressing strain was up to 10-fold lower than both the control and PGK overexpressing strains. It is proposed that expression of heterologous prochymosin imposes a greater energy drain on the host than overexpression of homologous PGK. This energetic drain may be a limiting factor in heterologous gene expression.     

Some characteristics of a raw starch digestion inhibitory factor fromAspergillus niger

Summary The effect of an inhibitory factor (IF) fromAspergillus niger 19 on raw starch digestion by pure glucoamylase I of blackAspergillus, pure glucoamylae ofRhizopus niveus, bacterial -amylase, fungal -amylase and various combination was investigated. The IF caused higher inhibition of raw starch hydrolysis by the combined action of glucoamylase and fungal -amylase than of hydrolysis by the individual enzymes. A protein moiety of IF might play an active part in this inhibition phenomenon. The IF was bound to starch granules, preventing hydrolysis by the enzymes, and caused decreased raw starch hydrolysis yields.     

Nematode infestation of fillets from Atlantic cod, Gadus morhua, off eastern Canada

The prevalence and intensity of larval nematodes in fillets of Atlantic cod, Gadus morhua, were examined and compared with similar data from a previous survey conducted about 30 yr ago. Anisakis simplex occurred more often in the nape of the fillet, whereas Pseudoterranova decipiens was the predominant species in napeless fillets. The results suggest an increase in both the prevalence and intensity of P. decipiens in fillets of cod, especially those originating from the Gulf of St. Lawrence and coastal Nova Scotia.     

Seasonal distribution of Aeromonas hydrophila in river water and isolation from river fish

The seasonal distribution of Aeromonas hydrophila in water and recovery rate from live river fish was investigated. The highest isolation rates of A. hydrophila occurred in water during the late winter followed by a progressive decline in density during the summer and monsoon seasons. The organism was recovered from fish throughout the period from which it was concluded that they form a reservoir which is unrelated to their density in water. The enterotoxigenicity of some environmental strains was tested in suckling mice and rabbit ileal loop.     

Hepatic membranolytic stability alteration by metalloporphins in rats.

The mothers of experimental neonates were administered excess bilirubin for a month, and the neonates were suffering from hyperbilirubinemia. The studies were conducted on the effect of excess bilirubin and metalloporphyrins on plasma membrane and mitochondrial membrane. We have isolated, separated, and estimated phospholipids, and also assayed the activity of phospholipase A2 from whole liver and mitochondrial and microsomal fractions. Excess of bilirubin administration decreased the total phospholipid level and inhibited the phospholipase A2 activity. Cr-PP (chromium protoporphyrin) induces the phospholipase A2 activity which is inhibited by simultaneous bilirubin administration. However, Zn-PP (zinc protoporphyrin) and Mn-PP (manganese protoporphyrin) showed a reverse pattern.