Kent Lai

Use of hyperspectral imaging to distinguish normal, precancerous, and cancerous cells†

The objective of the current study was to test the hypothesis that the cytologic diagnosis of cancer cells can be enhanced by the technique of hyperspectral imaging (HSI).

High-Throughput Screening for Human Galactokinase Inhibitors

Inherited deficiency of galactose-1-phosphate uridyltransferase (GALT) can result in a potentially lethal disorder called classic galactosemia. Although the neonatal lethality associated with this disease can be prevented through early diagnosis and a galactose-restricted diet, the lack of effective therapy continues to have consequences: developmental delay, neurological disorders, and premature ovarian failure are common sequelae in childhood and adulthood. Several lines of evidence indicate that an elevated level of galactose-1-phosphate (gal-1-p), the product of galactokinase (GALK), is a major, if not sole, pathogenic mechanism in patients with classic galactosemia. The authors hypothesize that elimination of gal-1-p production by inhibiting GALK will relieve GALT-deficient cells from galactose toxicity. To test this hypothesis, they obtained human GALK using a bacterial expression system. They developed a robust, miniaturized, high-throughput GALK assay (Z′ factor = 0.91) and used this assay to screen against libraries composed of 50,000 chemical compounds with diverse structural scaffolds. They selected 150 compounds that, at an average concentration of 33.3 µM, inhibited GALK activity in vitro more than 86.5% and with a reproducibility score of at least 0.7 for a confirmatory screen under identical experimental conditions. Of these 150 compounds, 34 were chosen for further characterization. Preliminary results indicated that these 34 compounds have potential to serve as leads to the development of more effective therapy of classic galactosemia. ( Journal of Biomolecular Screening 2008:415-423)

Molecular and Biochemical Characterization of Human Galactokinase and its small molecule inhibitors

Human galactokinase (GALK) is the first enzyme in the Leloir pathway, converting α-d-galactose into galactose-1-phosphate (Gal-1-P). Recently, there is increasing interest in targeting GALK as a novel therapy to ameliorate the disease manifestations in patients with Classic Galactosemia as it would, in combination with (ga-)lactose restriction reduce accumulation of Gal-1-P, a cytotoxic agent. Previously, we identified 34 small molecule compounds that inhibited GALK in vitro using experimental high-throughput screening. In order to isolate useful lead compounds, we characterized these hits with regards to their kinase selectivity profiles, potency and capability to reduce Gal-1-P accumulation in patient cell lines, and their modes of action. We found that the majority of these compounds had IC(50)s ranging from 0.7μM to 33.3μM. When tested against other members of the GHMP kinase family, three compounds (1, 4, and 24) selectively inhibited GALK with high potency. Through alignment of GALK and mevalonate kinase (MVK) crystal structures, we identified that eight amino acid residues and an L1 loop were different within the ATP-binding pockets of these two closely related kinases. By site-directed mutagenesis experiments, we identified one amino acid residue required for the inhibitory function of two of the three selective compounds. Based on these results, we generated binding models of these two compounds using a high-precision docking program. Compounds 4 and 24 inhibited GALK in a mixed model, while compound 1 exhibited parabolic competitive inhibition. Most importantly, using cells from galactosemic patients we found that selected compounds lowered Gal-1-P concentrations.

Subfertility and growth restriction in a new galactose-1 phosphate uridylyltransferase (GALT) - deficient mouse model

The first GalT gene knockout (KO) mouse model for Classic Galactosemia (OMIM 230400) accumulated some galactose and its metabolites upon galactose challenge, but was seemingly fertile and symptom free. Here we constructed a new GalT gene-trapped mouse model by injecting GalT gene-trapped mouse embryonic stem cells into blastocysts, which were later implanted into pseudo-pregnant females. High percentage GalT gene-trapped chimera obtained were used to generate heterozygous and subsequently, homozygous GalT gene-trapped mice. Biochemical assays confirmed total absence of galactose-1 phosphate uridylyltransferase (GALT) activity in the homozygotes. Although the homozygous GalT gene-trapped females could conceive and give birth when fed with normal chow, they had smaller litter size (P=0.02) and longer time-to-pregnancy (P=0.013) than their wild-type littermates. Follicle-stimulating hormone levels of the mutant female mice were not significantly different from the age-matched, wild-type females, but histological examination of the ovaries revealed fewer follicles in the homozygous mutants (P=0.007). Administration of a high-galactose (40% w/w) diet to lactating homozygous GalT gene-trapped females led to lethality in over 70% of the homozygous GalT gene-trapped pups before weaning. Cerebral edema, abnormal changes in the Purkinje and the outer granular cell layers of the cerebellum, as well as lower blood GSH/GSSG ratio were identified in the galactose-intoxicated pups. Finally, reduced growth was observed in GalT gene-trapped pups fed with normal chow and all pups fed with high-galactose (20% w/w) diet. This new mouse model presents several of the complications of Classic Galactosemia and will be useful to investigate pathogenesis and new therapies.

Black children deficient in galactose 1-phosphate uridyltransferase: correlation of activity and immunoreactive protein in erythrocytes and leukocytes.

A recent study found a high prevalence of a missense mutation (S135L) in the gene for galactose 1-phosphate uridyltransferase (GALT) in black children with galactosemia (J Pediatr 1996; 128:89-95). In the present study, GALT activity and GALT protein content were measured in erythrocytes and leukocytes of eight black and seven white galactosemic (GALT-deficient) children, for correlation with the presence of the S135L and Q188R (highly prevalent in white galactosemic children) missense mutations. The S135L mutation was found in 9 of 16 alleles of black children but not in white children; the Q188R mutation was found in 10 of 14 alleles examined in white galactosemic children and in 4 of 16 alleles in black galactosemic children. The GALT activity was near zero in the erythrocytes of white and black galactosemic children (0.26 +/- 0.28 vs 0.33 +/- 0.25 mumol/hr per gram of hemoglobin, respectively; p = 0.61) (normal 17 to 26 mumol/hr per gram), and no correlation of erythrocyte activity with genotype was observed. The GALT activity was higher in the leukocytes of black galactosemic children compared with white children (5 +/- 6 vs 1 +/- 2 mumol/hr per gram, respectively) (normal 172 to 374 mumol/hr per gram), but the difference was not statistically significant (p = 0.11). Analysis by genotype revealed that the two S135L homozygotes had much more leukocyte activity (9 and 17 mumol/hr per gram) than Q188R homozygotes or than all non-S135L allelic genotypes. Compound heterozygotes (S135L/G) had intermediate activity. The GALT protein was not detectable by Western blot in the erythrocytes of either white or black galactosemic children, as determined by antibodies specific for both C- and N-terminal sequences. The GALT protein was undetectable in the leukocytes of white galactosemic children, but leukocytes from black galactosemic children with the S135L mutation contained reduced but readily detectable GALT protein. Erythrocyte galactose 1-phosphate levels were significantly lower in galactosemic children with an S135L mutant allele (1.1 +/- 0.2 gm/dl) compared with children who had other mutations (3.1 +/- 0.9 mg/dl; p = 0.0001). The correlation of protein content data with activity levels in the blood cells suggests that the S135L missense mutation affects the stability of GALT protein to produce a deficiency state.

Prevention of a molecular misdiagnosis in galactosemia

Purpose: The polymerase chain reaction is generally used for mutational analysis of the galactose-1-phosphate uridyl transferase (GALT) gene in the diagnosis of galactosemia. This method is problematic when used in families of Ashkenazi Jewish descent.Methods: We amplified the GALT gene from leukocyte DNA followed by allele specific oligonucleotide hybridization, DNA sequencing and Southern Blot analysis to determine the mutant alleles causing galactosemia in a representative Jewish family.Results: The probands diagnosis of galactosemia was confirmed by high levels of erythrocyte galactose-1-phosphate, absence of erythrocyte GALT activity and impaired total body oxidation of galactose to expired CO2. Initial molecular analysis of GALT alleles in the family showed homozygosity for a K285N missense mutation in the proband, homozygosity for N314D in the mother and heterozygosity for N314D and K285N in the father. These results contradicted Mendelian logic. Southern blot hybridization with GALT cDNA proved the presence of a complex 5 kb GALT deletion in the proband and her mothers DNA enabling a corrected genotype.Conclusions: Since a deletion of the GALT gene is a common mutation causing galactosemia among Ashkenazim Jewish families, this deletion should be suspected and tested for by genomic hybridization or by using primers specific for the 5 kb deletion.

Transcriptomic analysis of Saccharomyces cerevisiae physiology in the context of galactose assimilation perturbations

Despite being extensively studied in various organisms due to scientific, industrial and medical interest, the galactose assimilation function and regulation, and especially its interaction with other parts of cellular physiology, have not been fully elucidated yet. The post-genomic era holistic techniques (omics) could assist towards this goal. In this paper, we holistically analyzed full-genome Saccharomyces cerevisiae transcriptional profiling data concerning its glucose- and galactose-grown wild-type (WT) physiology and its glucose-grown gal7-deficient (GAL7Delta) physiology, as these were obtained in the experiment described in Lai and Elsas (Biochem. Biophys. Res. Commun. 2000, 271, 392-400). The gal7 gene encodes for the second enzyme of the galactose assimilation, Leloir, pathway, and its deficiency in humans causes a potentially lethal disease, named classic galactosemia. Analysis of the galactose-grown compared to the glucose-grown WT physiology indicated a significant increase in the transcriptional expression of the ribosomal machinery and decrease in the transcriptional activity of the fatty acids beta-oxidation at the peroxisomes. Comparison of GAL7Delta to WT physiology in glucose indicated a significant transcriptional increase in the mitochondrial activity and the rate of catabolism of disaccharides, including sucrose, mannose and trehalose, towards amplified biosynthesis of the main cell wall components. Comparison with other physiological conditions indicated strong correlation between the glucose-grown GAL7Delta transcriptional physiology and the WT growth under glucose derepression conditions. Finally, the acquired results and the large number of still unknown genes that were related to the galactose assimilation regulation indicated the need for further, specifically designed, perturbations and integrated analyses of multiple levels of cellular function.

BIOCHEMICAL AND MOLECULAR BASIS FOR THE AFRICAN AMERICAN VARIANT OF GALACTOSEMIA. |[dagger]| 873

Early studies suggested that African American children (AA) with galactosemia developed fewer symptoms than white galactosemic children (W). The biochemical and molecular basis for this disparity has not been clarified. Galactosemia is caused by deficiency of galactose-1-phosphate uridyl transferase (GALT). In this study, GALT activity was measured in the RBC and WBC of 10 W and 8 AA. WBC activities were measured because WBC unlike RBC make GALT protein and may therefore more accurately reflect GALT activity in other tissues. GALT protein content was determined by Western blot. Mutational analysis of the gene was undertaken to determine the presence of Q188R (common mutation) and S135L (found only in AA) alleles. S135L was present in 9/16 AA alleles.