The Structural and Molecular Biology of Type I Galactosemia: Enzymology of Galactose 1-phosphate Uridylyltransferase

Reduced galactose 1-phosphate uridylyltransferase (GAIT) activity is associated with the genetic disease type 1 galactosemia. This results in an increase in the cellular concentration of galactose 1-phosphate. The accumulation of this toxic metabolite, combined with aberrant glycoprotein and glycolipid biosynthesis, is likely to be the major factor in molecular pathology. The mechanism of GAIT was established through classical enzymological methods to be a substituted enzyme in which the reaction with UDP-glucose results in the formation of a covalent, UMP-histidine adduct in the active site. The uridylated enzyme can then react with galactose 1-phosphate to form UDP-galactose. The structure of the enzyme from Escherichia coli reveals a homodimer containing one zinc (II) and one iron (11) ion per subunit. This enzymological and structural knowledge provides the basis for understanding the biochemistry of this critical step in the Leloir pathway. However, a high-resolution crystal structure of human GAIT is required to assist greater understanding of the effects of disease-associated mutations. (C) 2011 IUBMB IUBMB Life, 63(9): 694-700, 2011

Identification of a methylation hotspot in the death receptor Fas/CD95 in bladder cancer

We characterized Fas immunoreactivity, functionality and its role in the response to mitomycin-C (MMC) chemotherapy in vitro in cell lines and in vivo in bladder washings from 23 transitional cell carcinoma of the bladder (TCCB) patients, harvested prior to and during MMC intravesical treatment. Having established the importance of functional Fas, we investigated the methylation and exon 9 mutation as mechanisms of Fas silencing in TCCB. For the first time, we report p53 up-regulation in 9/14 and Fas up-regulation in 7/9 TCCB patients during intravesical MMC treatment. Fas immunoreactivity was strong in the TCCB cell line T24 and in 17/20 (85%) tumor samples from patients with advanced TCCB. T24 and HT1376 cells were resistant to MMC and recombinant Fas ligand, whilst RT4 cells were responsive to Fas ligand and MMC. Using RT4 cells as a model, siRNA targeting p53 significantly reduced MMC-induced p53 and Fas up-regulation and stable DN-FADD transfection decreased MMC-induced apoptosis, suggesting that functional Fas enhances chemotherapy responses in a p53-dependent manner. In HT1376 cells, 5-aza-2-deoxycytidine (12 µM) induced Fas immunoreactivity and reversed methylation at CpG site -548 within the Fas promoter. This site was methylated in 13/24 (54%) TCCB patient samples assessed using Methylation-Specific Polymerase Chain Reaction. There was no methylation at either the p53 enhancer region within the first intron or at the SP-1 binding region in the promoter and no mutation within exon 9 in tumor DNA extracted from 38 patients. Methylation at CpG site -548 is a potential target for demethylating drugs.

Diabetes-related adduct formation and retinopathy

The pathogenesis of diabetic retinopathy is complex, reflecting the array of systemic and tissue-specific metabolic abnormalities. A range of pathogenic pathways are directly linked to hyperglycaemia and dyslipidaemia, and the retina appears to be exquisitely sensitive to damage. Establishing the biochemical and molecular basis for this pathology remains an important research focus. This review concentrates on the formation of a range of protein adducts that form after exposure to modifying intermediates known to be elevated during diabetes. These so-called advanced glycation end products (AGEs) and advanced lipoxidation end products (ALEs) are thought to play an important role in the initiation and progression of diabetic retinopathy, and mechanisms leading to dysfunction and death of various retinal cells are becoming understood. Perspective is provided on AGE/ALE formation in the retina and the impact that such adducts have on retinal cell function. There will be emphasis placed on the role of the receptor for AGEs and how this may modulate retinal pathology, especially in relation to oxidative stress and inflammation. The review will conclude by discussion of strategies to inhibit AGE/ALE formation or harmful receptor interactions in order to prevent disease progression from the point of diabetes diagnosis to sight-threatening proliferative diabetic retinopathy and diabetic macular oedema.

POPULATION BIOLOGY OF HELIGMOSOMOIDES-POLYGYRUS (NEMATODA) IN THE WOOD MOUSE

1. This paper examines the interaction between the wood mouse, Apodemus sylvaticus L., and the intestinal nematode, Heligmosomoides polygyrus Dujardin, using data collected at Tollymore Park Forest, Co. Down, Northern Ireland, between November 1978 and July 1981.

Structural and Molecular Biology of Type I Galactosemia: Disease-associated Mutations

Type I galactosemia results from reduced galactose 1-phosphate uridylyltransferase (GALT) activity. Signs of disease include damage to the eyes, brain, liver, and ovaries. However, the exact nature and severity of the pathology depends on the mutation(s) in the patient's genes and his/her environment. Considerable enzymological and structural knowledge has been accumulated and this provides a basis to explain, at a biochemical level, impairment in the enzyme in the more than 230 disease-associated variants, which have been described. The most common variant, Q188R, occurs close to the active site and the dimer interface. The substitution probably disrupts both UDP-sugar binding and homodimer stability. Other alterations, for example K285N, occur close to the surface of the enzyme and most likely affect the folding and stability of the enzyme. There are a number of unanswered questions in the field, which require resolution. These include the possibility that the main enzymes of galactose metabolism form a supramolecular complex and the need for a high resolution crystal structure of human GALT. (C) 2011 IUBMB IUBMB Life, 63(11): 949-954, 2011