Characterization of the N314D allele of human galactose-1-phosphate uridylyltransferase using a yeast expression system.

Transferase-deficiency galactosemia is an inborn error of metabolism resulting from impairment of the enzyme galactose-1-phosphate uridylyltransferase (GALT), which normally catalyzes the second step of the Leloir pathway of galactose metabolism. Several recent studies have linked a previously reported substitution, N314D (asn to asp at position 314), with both the Duarte and Los Angeles (LA) variant alleles of GALT. While both variants demonstrate similar mobility shifts relative to the normal enzyme on isoelectric focusing (IEF) gels, one (Duarte) is associated with diminished activity, while the other (LA) is associated with greater than normal activity. Therefore, although the concordance rates between N314D and both of these phenotypes are compelling, the question remains as to whether N314D alone is sufficient to cause either or both variants. To address the question of precisely what properties of variant GALT can be attributed to the N314D substitution alone, we have modeled both the wildtype and N314D-GALT alleles in a previously defined yeast expression system, and characterized each with respect to activity, abundance, subunit interaction, and mobility on isoelectric focusing gels. Our results indicate that the N314D subunit dimerizes well both with wildtype GALT and with itself and that the N314D substitution is sufficient to confer the expected shift of IEF banding pattern associated with both the Duarte and LA variant proteins isolated from human cells. However, our results also suggest that N314D-GALT retains full specific activity, thereby calling into question the suggestion that N314D encodes the Duarte variant of GALT.

Identification and functional analysis of three distinct mutations in the human galactose-1-phosphate uridyltransferase gene associated with galactosemia in a single family.

We have identified three mutations associated with transferase-deficiency galactosemia in a three-generation family including affected members in two generations and have modeled all three mutations in a yeast-expression system. A sequence of pedigree, biochemical, and molecular analyses of the galactose-1-phosphate uridyltransferase (GALT) enzyme and genetic locus in both affected and carrier individuals revealed three distinct base substitutions in this family, two (Q188R and S135L) that had been reported previously and one (V151A) that was novel. Biochemical analyses of red-blood-cell lysates from the relevant family members suggested that each of these mutations was associated with dramatic impairment of GALT activity in these cells. While this observation was consistent with our previous findings concerning the Q188R mutation expressed both in humans and in a yeast-model system, it was at odds with a report by Reichardt and colleagues, indicating that in their COS cell-expression system the S135L substitution behaved as a neural polymorphism. To address this apparent paradox, as well as to investigate the functional significance of the newly identified V151A substitution, all three mutations were recreated by site-directed mutagenesis of the otherwise wild-type human GALT sequence and were expressed both individually and in the appropriate allelic combinations in a GALT-deficient strain of the yeast Saccharomyces cerevisiae.(ABSTRACT TRUNCATED AT 250 WORDS)

[Cytologic and morphometric features of mucosal cells of the cervical canal in the normal state and in pathology]. / Tsitologicheskie i morfometricheskie osobennosti kletok slizistoi obolochki tservikal'nogo kanala v norme i pri patologii.

Presents data on the morphology and function of the cervical canal mucosa, obtained with the use of colpocervicoscopy, as well as cytologic qualitative and quantitative characteristics of the glandular epithelium. Demonstrates high informative value and compatibility of the results obtained by different methods for the assessment of anaplasia and proliferation in the mucous endocervical cells, particularly in cases with the early evidence of the glandular epithelium malignant degeneration.