BBA - Molecular Basis of Disease (v.1537, #3)

SLC19A3 encodes a second thiamine transporter ThTr2 by Arun Rajgopal; Antoinette Edmondnson; I.David Goldman; Rongbao Zhao (175-178).
Recently, a new family of facilitative carriers has been cloned consisting of the reduced folate (SLC19A1) and the thiamine (SLC19A2) transporters. Despite a high level of sequence identity and similarity there is essentially no functional overlap between these carriers. The former transports folates and the latter thiamine. In this paper we describe the function of SLC19A3, another member of this transporter family most recently cloned, after transient transfection of the cDNA into HeLa cells. Uptake of [3H]thiamine, but not of methotrexate nor folic acid, was enhanced in SLC19A3 transfectants relative to vector control. Similarly, in the transfectants thiamine transport increased with an increase in pH with peak activity at pH ∼7.5. While [3H]thiamine uptake was markedly inhibited by nonlabeled thiamine it was not inhibited by several organic cations in 100-fold excess. Hence this carrier has a high degree of specificity for vitamin B1. The data indicate that SLC19A3 has the characteristics of SLC19A2 (ThTr1) and represents a second thiamine transporter (ThTr2) in this family of facilitative carriers.
Keywords: Thiamine transport; Thiamine transporter; SLC19A3 function; SLC19A family;

Islet amyloid polypeptide (IAPP, amylin) is secreted from pancreatic islet β-cells and converted to amyloid deposits in type 2 diabetes. Conversion from soluble monomer, IAPP 1–37, to β-sheet fibrils involves changes in the molecular conformation, cellular biochemistry and diabetes-related factors. In addition to the recognised amyloidogenic region, human IAPP (hIAPP) 20–29, the peptides human or rat IAPP 30–37 and 8–20, assume β-conformation and form fibrils. These three amyloidogenic regions of hIAPP can be modelled as a folding intermediate with an intramolecular β-sheet. A hypothesis is proposed for co-secretion of proIAPP with proinsulin in diabetes and formation of a ‘nidus’ adjacent to islet capillaries for subsequent accumulation of secreted IAPP to form the deposit. Although intracellular fibrils have been identified in experimental systems, extracellular deposition predominates in animal models and man. Extensive fibril accumulations replace islet cells. The molecular species of IAPP that is cytotoxic remains controversial. However, since fibrils form invaginations in cell membranes, small non-toxic IAPP fibrillar or amorphous accumulations could affect β-cell stimulus-secretion coupling. The level of production of hIAPP is important but not a primary factor in islet amyloidosis; there is little evidence for inappropriate IAPP hypersecretion in type 2 diabetes and amyloid formation is generated in transgenic mice overexpressing the gene for human IAPP only against a background of obesity. Animal models of islet amyloidosis suggest that diabetes is induced by the deposits whereas in man, fibril formation appears to result from diabetes-associated islet dysfunction. Islet secretory failure results from progressive amyloidosis which provides a target for new therapeutic interventions.
Keywords: Amyloid; Diabetes; Islet amyloid polypeptide; Islet; Fibril;

Risk of iron overload is decreased in beating heart coronary artery surgery compared to conventional bypass by Sharon Mumby; Tat W Koh; John R Pepper; John M.C Gutteridge (204-210).
Conventional cardiopulmonary bypass surgery (CCPB) increases the iron loading of plasma transferrin often to a state of plasma iron overload, with the presence of low molecular mass iron. Such iron is a potential risk factor for oxidative stress and microbial virulence. Here we assess ‘off-pump’ coronary artery surgery on the beating heart for changes in plasma iron chemistry. Seventeen patients undergoing cardiac surgery using the ‘Octopus’ myocardial wall stabilisation device were monitored at five time points for changes in plasma iron chemistry. This group was further divided into those (n=9) who had one- or two- (n=8) vessel grafts, and compared with eight patients undergoing conventional coronary artery surgery. Patients undergoing beating heart surgery had significantly lower levels of total plasma non-haem iron, and a decreased percentage saturation of their transferrin at all time points compared to conventional bypass patients. Plasma iron overload occurred in only one patient undergoing CCPB. Beating heart surgery appears to decrease red blood cell haemolysis, and tissue damage during the operative procedures and thereby significantly decreases the risk of plasma iron overload associated with conventional bypass.
Keywords: Heart bypass; Transferrin; Reactive iron species; Iron overload; Beating heart surgery; Oxidative stress;

There is no consensus regarding the results from in vivo and in vitro studies on the impact of chronic high insulin and/or high glucose exposure on acute insulin stimulation of glycogen synthase (GS) kinetic parameters in human skeletal muscle. The aim of this study was to evaluate the kinetic parameters of glycogen synthase activity in human myotube cultures at conditions of chronic high insulin combined or not with high glucose exposure, before and after a subsequent acute insulin stimulation. Acute insulin stimulation significantly increased the fractional activity (FV0.1) of GS, increased the sensitivity of GS to the allosteric activator glucose 6-phosphate (A 0.5) and increased the sensitivity of GS to its substrate UDPG (K m(0.1)) when myotubes were precultured at low insulin with/without high glucose conditions. However, this effect of acute insulin stimulation was abolished in myotubes precultured at high insulin with or without high glucose. Furthermore, we found significant correlations between the fractional velocities FV0.1 of GS and K m(0.1) (ρ=−0.72, P<0.0001), between FV0.1 and A 0.5 (ρ=−0.82, P<0.0001) and between K m(0.1) and A 0.5 values (ρ=0.71, P<0.0001). Our results show that chronic exposure of human myotubes to high insulin with or without high glucose did not affect the basal kinetic parameters but abolished the reactivity of GS to acute insulin stimulation. We suggest that insulin induced insulin resistance of GS is caused by a failure of acute insulin stimulation to decrease A 0.5 and K m(0.1) in human skeletal muscle.
Keywords: Cell culture; Skeletal muscle; Glycogen synthase; Insulin resistance; Kinetics; Human myotube;

Butylamino-demethoxy-hypocrellins and photodynamic therapy decreases human cancer in vitro and in vivo by Shangjie Xu; Shen Chen; Manhua Zhang; Tao Shen; Yupei Zhao; Ziwen Liu; Yuande Wu (222-232).
2-Butylamino-2-demethoxy-hypocrellin A (BAHA) and B (BAHB) are new photosensitizers synthesized by a mild reaction of hypocrellins and butylamine. In BAHA and BAHB, the peri-hydroxylated perylenequinone structure of the parent hypocrellins is preserved and the red absorption is enhanced distinctly. Electron paramagnetic resonance spin trapping measurements and 9,10-diphenylanthracene bleaching studies were used to investigate the photodynamic action of BAHA and BAHB in the presence of oxygen. Singlet oxygen (1O2) and superoxide anion radical (O⋅− 2) produced by illuminating BAHA and BAHB in aerobic solution have been observed. Compared with hypocrellin A and B, BAHA and BAHB primarily remained able to generate 1O2 and enhanced distinctly the O⋅− 2-generating abilities. The photodynamic action of BAHA and BAHB in the therapy of cancer was investigated in vitro and in vivo. Both in vitro and in vivo results revealed a significant decrease in cancer cell growth. Laser or dye alone had no effect, indicating that intratumor BAHA and laser therapy may prove useful in unresectable cancer.
Keywords: Hypocrellin; Photodynamic therapy; Pancreatic adenocarcinoma cancer; Electron paramagnetic resonance;

The effect of four mutations on the expression of iduronate-2-sulfatase in mucopolysaccharidosis type II by Gloria Bonuccelli; Paola Di Natale; Fabio Corsolini; Guglielmo Villani; Stefano Regis; Mirella Filocamo (233-238).
Mucopolysaccharidosis type II (Hunter syndrome; OMIM 309900) is a rare X-linked recessive lysosomal storage disorder caused by the deficiency of the enzyme iduronate-2-sulfatase (IDS; EC Different alterations at the IDS locus, mostly missense mutations, have been demonstrated, by expression study, as deleterious, causing significant consequences on the enzyme function or stability. In the present study we report on the results of the transient expression of the novel K347T, 533delTT, N265I and the already described 473delTCC (previously named ΔS117) mutations in the COS 7 cells proving their functional consequence on IDS activity. This type of information is potentially useful for genotype–phenotype correlation, prognosis and possible therapeutic intervention.
Keywords: Iduronate-2-sulfatase; Mucopolysaccharidosis type II; Hunter syndrome; Transient expression; COS cell; Western blot;

Variation in the promoter of the human hormone sensitive lipase gene shows gender specific effects on insulin and lipid levels: results from the Ely study by Philippa J Talmud; Jutta Palmen; Jian’an Luan; Dave Flavell; Christopher D Byrne; Dawn M Waterworth; Nicholas J Wareham (239-244).
We previously identified a hormone sensitive lipase (HSL) promoter variant, −60C>G, which in vitro exhibits 40% reduced promoter activity. In this study we examined the effect of the −60C>G on glycemic and lipid measures in the population based Ely study of metabolic function and insulin resistance in 218 middle-aged men and 276 middle-aged women. Adipose tissue HSL is the rate-limiting step in triglyceride lipolysis, generating free fatty acids for energy utilization. HSL is also expressed in pancreatic β-cells where its activity therefore may affect insulin secretion. In the women, carriers of the HSL −60G allele had significantly lower fasting insulin levels (P=0.0005) and a lower total area under the curve for insulin during the oral glucose tolerance test (P=0.005). There was no demonstrable association in men with these measures of insulin sensitivity but carriers of the −60G allele had significantly lower fasting non-esterified fatty acid (NEFA) levels (P=0.025) and higher low density lipoprotein cholesterol levels (P=0.02) than men who were non-carriers. This study provides additional evidence for a role for HSL in the development of insulin resistance, from which carriers of the −60G allele, associated here with markers of insulin sensitivity in women, and with lower NEFA levels in men, might be protected.
Keywords: Hormone sensitive lipase; Insulin resistance; Fasting insulin levels; Genetic polymorphism;

Author Index (245-246).

Contents Vol. 1537 (247-248).