Hexagonal phase with ordered acyl chains formed by a short chain asymmetric ceramide.

Ceramides constitute a group of lipids with usually high melting temperature that also favor negative curvature in membranes when mixed with other lipids. The short chain C10:0 ceramide is an asymmetric lipid which consists of an 18 carbon sphingosine base N-acylated with decanoic acid. According to high sensitivity differential scanning calorimetry, it shows a minor exothermic peak at 61°C and a main endothermic transition at 75°C. By small angle X-ray scattering and polarized light microscopy we found that, at temperatures below the main transition, the fully hydrated lipid dispersions are arranged in a tridimensional structure corresponding to an inverted hexagonal phase. Infrared spectroscopy and wide angle X-ray diffraction indicated that the acyl chains of ceramides exhibit a relatively high order in the hexagonal phase. As far as we know, this is the first report of a lipid hexagonal phase having highly ordered acyl chains. Molecular asymmetry due to the different length of the sphingosine and the N-acyl chains of C10:0 ceramide may explain why this novel phase is formed.

EBSD spatial resolution for detecting sigma phase in steels.

The spatial resolution of the electron backscatter diffraction signal is explored by Monte Carlo simulation for the sigma phase in steel at a typical instrumental set-up. In order to estimate the active volume corresponding to the diffracted electrons, the fraction of the backscattered electrons contributing to the diffraction signal was inferred by extrapolating the Kikuchi pattern contrast measured by other authors, as a function of the diffracted electron energy. In the resulting estimation, the contribution of the intrinsic incident beam size and the software capability to deconvolve patterns were included. A strong influence of the beam size on the lateral resolution was observed, resulting in 20nm for the aperture considered. For longitudinal and depth directions the resolutions obtained were 75nm and 16nm, respectively. The reliability of this last result is discussed in terms of the survey of the last large-angle deflection undergone by the backscattered electrons involved in the diffraction process. Bearing in mind the mean transversal resolution found, it was possible to detect small area grains of sigma phase by EBSD measurements, for a stabilized austenitic AISI 347 stainless steel under heat treatments, simulating post welding (40h at 600°C) and aging (284h at 484°C) effects-as usually occurring in nuclear reactor pressure vessels.

Day-restricted feeding during pregnancy and lactation programs glucose intolerance and impaired insulin secretion in male rat offspring.

AIM: The maternal environment during pregnancy and lactation plays a determining role in programming energy metabolism in offspring. Among a myriad of maternal factors, disruptions in the light/dark cycle during pregnancy can program glucose intolerance in offspring. Out-of-phase feeding has recently been reported to influence metabolism in adult humans and rodents; however, it is not known whether this environmental factor impacts offspring metabolism when applied during pregnancy and lactation. This study aims to determine whether maternal day-restricted feeding (DF) influences energy metabolism in offspring. METHODS: Pregnant and lactating Wistar rats were subjected to ad libitum (AL) or DF during pregnancy and lactation. The offspring born to the AL and DF dams were intra- and interfostered, which resulted in 4 group types. RESULTS: The male offspring born to and breastfed by the DF dams (DF/DF off) were glucose intolerant, but without parallel insulin resistance as adults. Experiments with isolated pancreatic islets demonstrated that the male DF/DF off rats had reduced insulin secretion with no parallel disruption in calcium handling. However, this reduction in insulin secretion was accompanied by increased miRNA-29a and miRNA34a expression and decreased syntaxin 1a protein levels. CONCLUSION: We conclude that out-of-phase feeding during pregnancy and lactation can lead to glucose intolerance in male offspring, which is caused by a disruption in insulin secretion capacity. This metabolic programming is possibly caused by mechanisms dependent on miRNA modulation of syntaxin 1a.

MCT1 and MCT4 kinetic of mRNA expression in different tissues after aerobic exercise at maximal lactate steady state workload.

We evaluate the mRNA expression of monocarboxylate transporters 1 and 4 (MCT1 and MCT4) in skeletal muscle (soleus, red and white gastrocnemius), heart and liver tissues in mice submitted to a single bout of swimming exercise at the maximal lactate steady state workload (MLSSw). After 72 h of MLSS test, the animals were submitted to a swimming exercise session for 25 min at individual MLSSw. Tissues and muscle samples were obtained at rest (control, n=5), immediately (n=5), 5 h (n=5) and 10 h (n=5) after exercise for determination of the MCT1 and MCT4 mRNA expression (RT-PCR). The MCT1 mRNA expression in liver increased after 10 h in relation to the control, immediate and 5 h groups, but the MCT4 remained unchanged. The MCT1 mRNA expression in heart increased by 31 % after 10 h when compared to immediate, but no differences were observed in relation to the control group. No significant differences were observed for red gastrocnemius in MCT1 and MCT4 mRNA expression. However, white gastrocnemius increased MCT1 mRNA expression immediately when compared to rest, 5 and 10 h test groups. In soleus muscle, the MCT1 mRNA expression increased immediately, 5 and 10 h after exercise when compared to the control. In relation to MCT4 mRNA expression, the soleus increased immediately and 10 h after acute exercise when compared to the control group. The soleus, liver and heart were the main tissues that showed improved the MCT1 mRNA expression, indicating its important role in controlling MLSS concentration in mice.

Effects of melatonin on DNA damage induced by cyclophosphamide in rats.

The antioxidant and free radical scavenger properties of melatonin have been well described in the literature. In this study, our objective was to determine the protective effect of the pineal gland hormone against the DNA damage induced by cyclophosphamide (CP), an anti-tumor agent that is widely applied in clinical practice. DNA damage was induced in rats by a single intraperitoneal injection of CP (20 or 50 mg/kg). Animals received melatonin during the dark period for 15 days (1 mg/kg in the drinking water). Rat bone marrow cells were used for the determination of chromosomal aberrations and of formamidopyrimidine DNA glycosylase enzyme (Fpg)-sensitive sites by the comet technique and of Xpf mRNA expression by qRT-PCR. The number (mean ± SE) of chromosomal aberrations in pinealectomized (PINX) animals treated with melatonin and CP (2.50 ± 0.50/100 cells) was lower than that obtained for PINX animals injected with CP (12 ± 1.8/100 cells), thus showing a reduction of 85.8% in the number of chromosomal aberrations. This melatonin-mediated protection was also observed when oxidative lesions were analyzed by the Fpg-sensitive assay, both 24 and 48 h after CP administration. The expression of Xpf mRNA, which is involved in the DNA nucleotide excision repair machinery, was up-regulated by melatonin. The results indicate that melatonin is able to protect bone marrow cells by completely blocking CP-induced chromosome aberrations. Therefore, melatonin administration could be an alternative and effective treatment during chemotherapy.

Effects of melatonin on DNA damage induced by cyclophosphamide in rats

The antioxidant and free radical scavenger properties of melatonin have been well described in the literature. In this study, our objective was to determine the protective effect of the pineal gland hormone against the DNA damage induced by cyclophosphamide (CP), an anti-tumor agent that is widely applied in clinical practice. DNA damage was induced in rats by a single intraperitoneal injection of CP (20 or 50 mg/kg). Animals received melatonin during the dark period for 15 days (1 mg/kg in the drinking water). Rat bone marrow cells were used for the determination of chromosomal aberrations and of formamidopyrimidine DNA glycosylase enzyme (Fpg)-sensitive sites by the comet technique and of Xpf mRNA expression by qRT-PCR. The number (mean ± SE) of chromosomal aberrations in pinealectomized (PINX) animals treated with melatonin and CP (2.50 ± 0.50/100 cells) was lower than that obtained for PINX animals injected with CP (12 ± 1.8/100 cells), thus showing a reduction of 85.8% in the number of chromosomal aberrations. This melatonin-mediated protection was also observed when oxidative lesions were analyzed by the Fpg-sensitive assay, both 24 and 48 h after CP administration. The expression of Xpf mRNA, which is involved in the DNA nucleotide excision repair machinery, was up-regulated by melatonin. The results indicate that melatonin is able to protect bone marrow cells by completely blocking CP-induced chromosome aberrations. Therefore, melatonin administration could be an alternative and effective treatment during chemotherapy.

Effect of triiodothyronine on the maxilla and masseter muscles of the rat stomatognathic system

The maxilla and masseter muscles are components of the stomatognathic system involved in chewing, which is frequently affected by physical forces such as gravity, and by dental, orthodontic and orthopedic procedures. Thyroid hormones (TH) are known to regulate the expression of genes that control bone mass and the oxidative properties of muscles; however, little is known about the effects of TH on the stomatognathic system. This study investigated this issue by evaluating: i) osteoprotegerin (OPG) and osteopontine (OPN) mRNA expression in the maxilla and ii) myoglobin (Mb) mRNA and protein expression, as well as fiber composition of the masseter. Male Wistar rats (~250 g) were divided into thyroidectomized (Tx) and sham-operated (SO) groups (N = 24/group) treated with T3 or saline (0.9 percent) for 15 days. Thyroidectomy increased OPG (~40 percent) and OPN (~75 percent) mRNA expression, while T3 treatment reduced OPG (~40 percent) and OPN (~75 percent) in Tx, and both (~50 percent) in SO rats. Masseter Mb mRNA expression and fiber type composition remained unchanged, despite the induction of hypo- and hyperthyroidism. However, Mb content was decreased in Tx rats even after T3 treatment. Since OPG and OPN are key proteins involved in the osteoclastogenesis inhibition and bone mineralization, respectively, and that Mb functions as a muscle store of O2 allowing muscles to be more resistant to fatigue, the present data indicate that TH also interfere with maxilla remodeling and the oxidative properties of the masseter, influencing the function of the stomatognathic system, which may require attention during dental, orthodontic and orthopedic procedures in patients with thyroid diseases.

Effect of triiodothyronine on the maxilla and masseter muscles of the rat stomatognathic system.

The maxilla and masseter muscles are components of the stomatognathic system involved in chewing, which is frequently affected by physical forces such as gravity, and by dental, orthodontic and orthopedic procedures. Thyroid hormones (TH) are known to regulate the expression of genes that control bone mass and the oxidative properties of muscles; however, little is known about the effects of TH on the stomatognathic system. This study investigated this issue by evaluating: i) osteoprotegerin (OPG) and osteopontine (OPN) mRNA expression in the maxilla and ii) myoglobin (Mb) mRNA and protein expression, as well as fiber composition of the masseter. Male Wistar rats (~250 g) were divided into thyroidectomized (Tx) and sham-operated (SO) groups (N = 24/group) treated with T3 or saline (0.9%) for 15 days. Thyroidectomy increased OPG (~40%) and OPN (~75%) mRNA expression, while T3 treatment reduced OPG (~40%) and OPN (~75%) in Tx, and both (~50%) in SO rats. Masseter Mb mRNA expression and fiber type composition remained unchanged, despite the induction of hypo- and hyperthyroidism. However, Mb content was decreased in Tx rats even after T3 treatment. Since OPG and OPN are key proteins involved in the osteoclastogenesis inhibition and bone mineralization, respectively, and that Mb functions as a muscle store of O2 allowing muscles to be more resistant to fatigue, the present data indicate that TH also interfere with maxilla remodeling and the oxidative properties of the masseter, influencing the function of the stomatognathic system, which may require attention during dental, orthodontic and orthopedic procedures in patients with thyroid diseases.

Peri-implantitis fibroblasts respond to host immune factor C1q.

BACKGROUND AND OBJECTIVE: Current therapies for peri-implantitis apply the same clinical protocols as those used for the treatment of periodontitis; however, outcomes remain unpredictable. We hypothesized that resident fibroblasts of the peri-implantitis stroma and periodontitis stroma differ in their phenotype and response to host immune factors. Fibroblasts are highly heterogeneous and comprise discrete subtypes with the potential of modulating inflammatory activities. The aim of the present study was to characterize the expression of receptors for complement C1q of innate immunity on human peri-implantitis fibroblasts and investigate effects of C1q on the proinflammatory properties of the cells. MATERIAL AND METHODS: Fibroblasts were cultured from gingival tissues exhibiting peri-implantitis and periodontitis, and from healthy gingivae as a control. Expression of C1q receptors for the collagen (cC1qR) and globular domains (gC1qR) of the protein was determined by flow cytofluorometric analysis (FITC) of specific antibodies bound to the surface of the cells. Secretion of C1q-inducible proinflammatory mediators was quantified after 24 h incubation using array-based ELISAs. RESULTS: The percentage of fibroblasts FITC-positive for cC1qR was 67, 75 and 12% in peri-implantitis, healthy and periodontitis cultures, respectively, whereas the percentage of gC1qR FITC-positive fibroblasts was 5, 3 and 59%, respectively. The C1q interactions with peri-implantitis and healthy fibroblasts increased secretion of the chemokines interleukin-6 and interleukin-8 twofold, and monocyte chemoattractant protein-1 fourfold over baseline values, whereas periodontitis fibroblasts were unresponsive. Complement C1q increased levels of vascular endothelial growth factor sevenfold and transforming growth factor-ß1 12-fold over baseline values in peri-implantitis cultures, only. CONCLUSIONS: Peri-implantitis fibroblasts differ from periodontitis fibroblasts in phenotypic expression of cC1qR and function, and from healthy fibroblasts in proinflammatory, angiogenic and fibrogenic function. Peri-implantitis fibroblasts may represent a novel subtype.

Association of NAD(P)H oxidase with glucose-induced insulin secretion by pancreatic beta-cells.

We previously described the presence of nicotinamide adenine dinucleotide phosphate reduced form [NAD(P)H]oxidase components in pancreatic beta-cells and its activation by glucose, palmitic acid, and proinflammatory cytokines. In the present study, the importance of the NAD(P)H oxidase complex for pancreatic beta-cell function was examined. Rat pancreatic islets were incubated in the presence of glucose plus diphenyleneiodonium, a NAD(P)H oxidase inhibitor, for 1 h or with the antisense oligonucleotide for p47(PHOX) during 24 h. Reactive oxygen species (ROS) production was determined by a fluorescence assay using 2,7-dichlorodihydrofluorescein diacetate. Insulin secretion, intracellular calcium responses, [U-(14)C]glucose oxidation, and expression of glucose transporter-2, glucokinase and insulin genes were examined. Antisense oligonucleotide reduced p47(PHOX) expression [an important NAD(P)H oxidase cytosolic subunit] and similarly to diphenyleneiodonium also blunted the enzyme activity as indicated by reduction of ROS production. Suppression of NAD(P)H oxidase activity had an inhibitory effect on intracellular calcium responses to glucose and glucose-stimulated insulin secretion by isolated islets. NAD(P)H oxidase inhibition also reduced glucose oxidation and gene expression of glucose transporter-2 and glucokinase. These findings indicate that NAD(P)H oxidase activation plays an important role for ROS production by pancreatic beta-cells during glucose-stimulated insulin secretion. The importance of this enzyme complex for the beta-cell metabolism and the machinery involved in insulin secretion were also shown.

Na(+) -glucose transporter-2 messenger ribonucleic acid expression in kidney of diabetic rats correlates with glycemic levels: involvement of hepatocyte nuclear factor-1alpha expression and activity.

Mutations in Na(+)-glucose transporters (SGLT)-2 and hepatocyte nuclear factor (HNF)-1alpha genes have been related to renal glycosuria and maturity-onset diabetes of the young 3, respectively. However, the expression of these genes have not been investigated in type 1 and type 2 diabetes. Here in kidney of diabetic rats, we tested the hypotheses that SGLT2 mRNA expression is altered; HNF-1alpha is involved in this regulation; and glycemic homeostasis is a related mechanism. The in vivo binding of HNF-1alpha into the SGLT2 promoter region in renal cortex was confirmed by chromatin immunoprecipitation assay. SGLT2 and HNF-1alpha mRNA expression (by Northern and RT-PCR analysis) and HNF-1 binding activity of nuclear proteins (by EMSA) were investigated in diabetic rats and treated or not with insulin or phlorizin (an inhibitor of SGLT2). Results showed that diabetes increases SGLT2 and HNF-1alpha mRNA expression (~50%) and binding of nuclear proteins to a HNF-1 consensus motif (~100%). Six days of insulin or phlorizin treatment restores these parameters to nondiabetic-rat levels. Moreover, both treatments similarly reduced glycemia, despite the differences in plasma insulin and urinary glucose concentrations, highlighting the plasma glucose levels as involved in the observed modulations. This study shows that SGLT2 mRNA expression and HNF-1alpha expression and activity correlate positively in kidney of diabetic rats. It also shows that diabetes-induced changes are reversed by lowering glycemia, independently of insulinemia. Our demonstration that HNF-1alpha binds DNA that encodes SGLT2 supports the hypothesis that HNF-1alpha, as a modulator of SGLT2 expression, may be involved in diabetic kidney disease.

Differential chemokine response of fibroblast subtypes to complement C1q.

BACKGROUND AND OBJECTIVE: The pathogenesis of periodontitis includes an inappropriate activation of the classical complement cascade (C') with accumulation of inflammatory C' products in fluids and tissues. Our hypothesis is that in vivo the C' product, C1q, may act as a regulatory component of the innate immune response of distinct matrix fibroblasts to the inflammatory environment. This study analyzed the C1q induction of pro-inflammatory cytokine secretion in fibroblast subtypes derived from distinct periodontal tissues, and identified a mechanism of the cell response. MATERIAL AND METHODS: Primary human gingival fibroblast, periodontal ligament fibroblast, and granulation tissue fibroblast cultures were treated for 24 h with C1q. Protein arrays assessed the secretory profile of constitutive and C1q-inducible pro-inflammatory cytokines, and enzyme-linked immunosorbent assays were used to quantify the kinetics of each inducible cytokine. RESULTS: Granulation tissue fibroblast cultures were unresponsive to C1q challenge. In contrast, periodontal ligament fibroblasts responded with a release of monocyte chemoattractant protein (MCP)-1, interleukin-6, interleukin-8, and macrophage inflammatory protein (MIP)-1beta higher than the basal level by 8.2-, 7.0-, 3.8-, and 7.2-fold, respectively. Human gingival fibroblast cultures increased secretion of these chemokines by 5.2-, 4.5-, 3.0-, and 9.8-fold, respectively. Inhibitor studies revealed that C1q-inducible release of chemokines by the human gingival fibroblast and periodontal ligament cultures was contingent upon p38 mitogen-activated protein kinase activity. CONCLUSION: The ability of C1q to stimulate secretion of pro-inflammatory chemokines depends upon which specific fibroblast subtype is involved. Targeting C1q-activated intracellular signaling pathways may be an effective means to inhibit the production of chemokines that promote inflammatory cell infiltration into gingival and periodontal ligament tissues.

Glucose, palmitate and pro-inflammatory cytokines modulate production and activity of a phagocyte-like NADPH oxidase in rat pancreatic islets and a clonal beta cell line.

AIMS/HYPOTHESIS: Acute or chronic exposure of beta cells to glucose, palmitic acid or pro-inflammatory cytokines will result in increased production of the p47(phox) component of the NADPH oxidase and subsequent production of reactive oxygen species (ROS). METHODS: Rat pancreatic islets or clonal rat BRIN BD11 beta cells were incubated in the presence of glucose, palmitic acid or pro-inflammatory cytokines for periods between 1 and 24 h. p47(phox) production was determined by western blotting. ROS production was determined by spectrophotometric nitroblue tetrazolium or fluorescence-based hydroethidine assays. RESULTS: Incubation for 24 h in 0.1 mmol/l palmitic acid or a pro-inflammatory cytokine cocktail increased p47(phox) protein production by 1.5-fold or by 1.75-fold, respectively, in the BRIN BD11 beta cell line. In the presence of 16.7 mmol/l glucose protein production of p47(phox) was increased by 1.7-fold in isolated rat islets after 1 h, while in the presence of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1beta it was increased by 1.4-fold or 1.8-fold, respectively. However, palmitic acid or IL-1beta-dependent production was reduced after 24 h. Islet ROS production was significantly increased after incubation in elevated glucose for 1 h and was completely abolished by addition of diphenylene iodonium, an inhibitor of NADPH oxidase or by the oligonucleotide anti-p47(phox). Addition of 0.1 mmol/l palmitic acid or 5 ng/ml IL-1beta plus 5.6 mmol/l glucose also resulted in a significant increase in islet ROS production after 1 h, which was partially attenuated by diphenylene iodonium or the protein kinase C inhibitor GF109203X. However, ROS production was reduced after 24 h incubation. CONCLUSIONS/INTERPRETATION: NADPH oxidase may play a key role in normal beta cell physiology, but under specific conditions may also contribute to beta cell demise.

C1q arrests the cell cycle progression of fibroblasts in G(1) phase: role of the cAMP/PKA-I pathway.

C1q may participate in the loss of connective tissue occurring in chronic inflammatory lesions. The hypothesis of a detrimental role of C1q on cell proliferation was tested on primary cultures of human fibroblasts (HFs). C1q suppressed the DNA synthesis of HF in response to platelet-derived growth factor (PDGF) with an IC(50) of 20 microg/ml, and blocked 78% of the cycling cells in G(1) phase. The C1q block did not involve production of inhibitory prostaglandin by the cells. Given that C1q elicits signals of the adenylyl cyclase pathway in HF, we examined cAMP-dependent mechanisms to understand how C1q inhibited the PDGF response. Whereas the C1q block was enhanced by agonist dibutyryl-adenosine 3', 5'-cyclic mono-phosphate (db-cAMP), antagonist adenosine 3', 5'-cyclic monophosphorotioate triethylammonium salt (Rp-cAMP) minimized it. C1q increased the level of cAMP-dependent protein kinase I (PKA-I) 4.5-fold, without altering the activation of the extracellular-regulated protein kinase (ERK) pathway. These results demonstrate that the interactions of C1q with HF cause growth arrest at the G(1) phase through mechanisms associated with a PKA-I dependent pathway.

The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis.

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Tetracaine stimulates insulin secretion through the mobilization of Ca2+ from thapsigargin- and IP3-insensitive Ca2+ reservoir in pancreatic beta-cells.

The effect of tetracaine on 45Ca efflux, cytoplasmic Ca2+ concentration [Ca2+]i, and insulin secretion in isolated pancreatic islets and beta-cells was studied. In the absence of external Ca2+, tetracaine (0.1-2.0 mM) increased the 45Ca efflux from isolated islets in a dose-dependentOFF efflux caused by 50 mM K+ or by the association of carbachol (0.2 mM) and 50 mM K+. Tetracaine permanently increased the [Ca2+]i in isolated beta-cells in Ca2+-free medium enriched with 2.8 mM glucose and 25 microM D-600 (methoxiverapamil). This effect was also observed in the presence of 10 mM caffeine or 1 microM thapsigargin. In the presence of 16.7 mM glucose, tetracaine transiently increased the insulin secretion from islets perfused in the absence and presence of external Ca2+. These data indicate that tetracaine mobilises Ca2+ from a thapsigargin-insensitive store and stimulates insulin secretion in the absence of extracellular Ca2+. The increase in 45Ca efflux caused by high concentrations of K+ and by carbachol indicates that tetracaine did not interfere with a cation or inositol triphosphate sensitive Ca2+ pool in beta-cells.

Molecular heterogeneity of the A3 subgroup.

The molecular characterization of the subgroup A3 remains unclear. Four unrelated A3 blood donors were studied. Family studies were possible in three of them. The A3 subgroup was defined by immunohaematological evaluation with four different commercially available serums. Exons VI and VII of the ABO gene, responsible for 91% of the catalytic active part of the glycosyltransferase, were amplified and subjected to direct sequencing. The results in all samples showed heterozygosity for the G261 deletion. In the A3 allele, the following associations were found: C467T mutation and 1060C deletion in one A3 blood donor and in another G829A and 1060C. In one case, only the 1060C deletion was demonstrated in the A3 allele. One blood donor presented the T646A and the G829A mutations in homozygosity. It was concluded that the A3 blood group is very heterogeneous at the molecular level.