A test-retest multisite reproducibility study of cardiovascular four-dimensional flow MRI without respiratory gating.

AIM: To systematically investigate the multisite reproducibility, test-retest reliability, and observer variability of non-respiratory-gated four-dimensional (4D) flow magnetic resonance imaging (MRI) in the thoracic great vessels for the assessment of blood flow and peak velocity. MATERIALS AND METHODS: Electrocardiogram (ECG)-gated 4D flow MRI data were acquired without respiratory gating in 10 healthy volunteers. To analyse multisite reproducibility, 4D flow was scanned at three different sites using a 3 T GE MRI machine with identical protocols for the group of participants. In addition, to evaluate test-retest reliability, the same volunteers were scanned in each centre during a second visit. Data analysis included calculation of peak systolic velocity and time-resolved and total flow of both the ascending aorta and pulmonary artery. Two observers conducted the above measurements to assess the interobserver variability. RESULTS: Multisite, test-retest, interobserver agreement were good for the calculation of total flow and peak systolic velocity (mean differences <10% of the average flow parameter). CONCLUSION: Non-respiratory-gated 4D MRI-based assessment of aortic and pulmonary blood flow can be performed with good reproducibility. It may facilitate the potential clinical application of this technique.

Abnormal spontaneous brain activity in type 2 diabetic retinopathy revealed by amplitude of low-frequency fluctuations: a resting-state fMRI study.

AIM: To explore the altered spontaneous cerebral activity patterns and impaired functional regions in patients with diabetic retinopathy (DR) using resting-state functional magnetic resonance imaging (rs-fMRI) based on the amplitude of low-frequency fluctuations (ALFF) algorithm. MATERIALS AND METHODS: Twenty-one patients with DR (mean age, 54.9±9.9 years; 11 females) and 17 healthy control subjects (54.8±5.7 years; 9 females) were prospectively studied. The DR patients underwent laboratory tests. All individuals underwent a neuropsychological test. The differences in the ALFF values between the two groups were compared. The relationships between ALFF values and clinical measurements were analysed using a multiple-factor analysis. RESULTS: Compared to the controls, the DR group showed significantly increased ALFF values in the bilateral occipital gyrus, right lingual gyrus, and precuneus, and decreased values in the right posterior/anterior cerebellar lobe and the parahippocampal, fusiform, superior temporal, inferior parietal, and angular gyrus. Furthermore, the Montreal Cognitive Assessment (MoCA) scores were negatively correlated with decreased ALFF values in the right occipital lobe of the DR group, while increased ALFF values in the right precuneus and lingual gyrus were found to be positively correlated with glycosylated haemoglobin (HbA1c) levels. CONCLUSIONS: Patients with DR showed spontaneous cerebral activity abnormalities in many cerebral regions that were associated with cognitive impairments. HbA1c levels altered spontaneous cerebral activity in DR patients.

Evaluation of nasal patency by visual analogue scale/nasal obstruction symptom evaluation questionnaires and anterior active rhinomanometry after septoplasty: a retrospective one-year follow-up cohort study.

OBJECTIVE: To assess the efficacy of septoplasty and the correlation between the subjective evaluations of a visual analogue scale (VAS) and the Nasal Obstruction Symptom Evaluation (NOSE) questionnaire and active anterior rhinomanometry of the nasal airway after septoplasty. DESIGN: A retrospective, individual cohort study. SETTING: Ear, Nose and Throat Department, Taipei City Hospital, Taipei, Taiwan. PARTICIPANTS: Fifty patients with chronic nasal obstruction were enrolled in the study. All 50 patients underwent septoplasty because of nasal septal deviation. Another 28 patients without nasal symptoms served as controls. MAIN OUTCOME MEASURES: VAS, NOSE and active anterior rhinomanometry were used to measure the sensation of nasal obstruction. All measurements were performed in both groups preoperatively and then repeated on three postoperative visits (3, 6 and 12 months). RESULTS: The mean VAS score, NOSE score and the nasal resistance in the narrow side of the nose in the study group showed reduced symptoms at 3, 6 and 12 months postoperatively compared with the respective preoperative measurements (P < 0.001, all). The VAS and NOSE scores did not significantly correlate with total nasal resistance preoperatively or postoperatively. The VAS and nasal resistance in the obstructed nasal cavity correlated significantly preoperatively (P < 0.05), but not postoperatively. CONCLUSIONS: The subjective and objective symptoms of nasal obstruction had improved 1 year after septoplasty. A significant correlation between VAS scores and nasal resistance in the narrow side of the nose was found before surgery. The subjective and objective measurements of nasal obstruction lacked significant correlation postoperatively.

Glucose-6-phosphatase dependent substrate transport in the glycogen storage disease type-1a mouse.

Glycogen storage disease type 1a (GSD-1a) is caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. A G6Pase knockout mouse which mimics the pathophysiology of human GSD-1a patients was created to understand the pathogenesis of this disorder, to delineate the mechanisms of G6Pase catalysis, and to develop future therapeutic approaches. By examining G6Pase in the liver and kidney, the primary gluconeogenic tissues, we demonstrate that glucose-6-P transport and hydrolysis are performed by separate proteins which are tightly coupled. We propose a modified translocase catalytic unit model for G6Pase catalysis.

Mutations in the glucose-6-phosphatase gene that cause glycogen storage disease type 1a.

Glycogen storage disease (GSD) type 1a is caused by the deficiency of D-glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. Despite both a high incidence and morbidity, the molecular mechanisms underlying this deficiency have eluded characterization. In the present study, the molecular and biochemical characterization of the human G6Pase complementary DNA, its gene, and the expressed protein, which is indistinguishable from human microsomal G6Pase, are reported. Several mutations in the G6Pase gene of affected individuals that completely inactivate the enzyme have been identified. These results establish the molecular basis of this disease and open the way for future gene therapy.

Angiotensin mediates renal fibrosis in the nephropathy of glycogen storage disease type Ia.

Patients with glycogen storage disease type Ia (GSD-Ia) develop renal disease of unknown etiology despite intensive dietary therapies. This renal disease shares many clinical and pathological similarities to diabetic nephropathy. We studied the expression of angiotensinogen, angiotensin type 1 receptor, transforming growth factor-beta1, and connective tissue growth factor in mice with GSD-Ia and found them to be elevated compared to controls. While increased renal expression of angiotensinogen was evident in 2-week-old mice with GSD-Ia, the renal expression of transforming growth factor-beta and connective tissue growth factor did not increase for another week; consistent with upregulation of these factors by angiotensin II. The expression of fibronectin and collagens I, III, and IV was also elevated in the kidneys of mice with GSD-Ia, compared to controls. Renal fibrosis was characterized by a marked increase in the synthesis and deposition of extracellular matrix proteins in the renal cortex and histological abnormalities including tubular basement membrane thickening, tubular atrophy, tubular dilation, and multifocal interstitial fibrosis. Our results suggest that activation of the angiotensin system has an important role in the pathophysiology of renal disease in patients with GSD-Ia.

Identification of mutations in the gene for glucose-6-phosphatase, the enzyme deficient in glycogen storage disease type 1a.

Glycogen storage disease (GSD) type 1a is an autosomal recessive inborn error of metabolism caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase), the key enzyme in glucose homeostasis. Southern blot hybridization analysis using a panel of human-hamster hybrids showed that human G6Pase is a single-copy gene located on chromosome 17. To correlate specific defects with clinical manifestations of this disorder, we identified mutations in the G6Pase gene of GSD type 1a patients. In the G6Pase gene of a compound heterozygous patient (LLP), two mutations in exon 2 of one allele and exon 5 of the other allele were identified. The exon 2 mutation converts an arginine at codon 83 to a cysteine (R83C). This mutation, previously identified by us in another GSD type 1a patient, was shown to have no detectable phosphohydrolase activity. The exon 5 mutation in the G6Pase gene of LLP converts a glutamine codon at 347 to a stop (Q347SP). This Q347SP mutation was also detected in all exon 5 subclones (five for each patient) of two homozygous patients, KB and CB, siblings of the same parents. The predicted Q347SP mutant G6Pase is a truncated protein of 346 amino acids, 11 amino acids shorter than the wild type G6Pase of 357 residues. Site-directed mutagenesis and transient expression assays demonstrated that G6Pase-Q347SP was devoid of G6Pase activity. G6Pase is an endoplasmic reticulum (ER) membrane-associated protein containing an ER retention signal, two lysines (KK), located at residues 354 and 355. We showed that the G6Pase-K355SP mutant containing a lysine-355 to stop codon mutation is enzymatically active. Our data demonstrate that the ER protein retention signal in human G6Pase is not essential for activity. However, residues 347-354 may be required for optimal G6Pase catalysis.

In vitro studies of platelet adhesion, activation, and protein adsorption on curcumin-eluting biodegradable stent materials.

A major complication of coronary stenting is in-stent restenosis (ISR) due to thrombus formation. We hypothesized that locally released curcumin from coronary stent surface would inhibit ISR due to thrombus formation because of antithrombosis of curcumin. In the present work, curcumin-eluting polylactic acid-co-glycolic acid (PLGA) films were fabricated and their properties in vitro were investigated. The in vitro platelet adhesion and activation, as well as protein adsorption on curcumin-loading PLGA films were investigated to evaluate the blood compatibility of curcumin-eluting films. The structure of curcumin-eluting PLGA film and control was examined by Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy indicating that the peaks of curcumin did not shift in curcumin-eluting films. The results of contact angle and surface free energy indicated that loading curcumin in PLGA would make PLGA become more hydrophilic, which contributed to the increase of polar fraction of surface free energy. With the increase of curcumin in films, platelets adhering to the curcumin-eluting films decreased significantly. The number of activation platelets decreased after incorporating curcumin in PLGA films. Loading curcumin in PLGA film can markedly reduce the fibrinogen adsorption. All results indicated that incorporating curcumin in PLGA film can improve the blood compatibility of PLGA films. It can be used to fabricate drug-eluting stent to prevent thrombosis formation.

Improved blood compatibility of rapamycin-eluting stent by incorporating curcumin.

This paper dealt with improving the blood compatibility of the rapamycin-eluting stent by incorporating curcumin. The rapamycin- and rapamycin/curcumin-loaded PLGA (poly(d,l-lactic acid-co-glycolic acid)) coatings were fabricated onto the surface of the stainless steel stents using an ultrasonic atomization spray method. The structure of the coating films was characterized by Fourier transform infrared spectroscopy (FTIR). The optical microscopy and scanning electron microscopy (SEM) images of the drug-eluting stents indicated that the surface of all drug-eluting stents was very smooth and uniform, and there were not webbings and "bridges" between struts. There were not any cracks and delaminations on stent surface after expanded by the angioplasty balloon. The in vitro platelet adhesion and activation were investigated by static platelet adhesion test and GMP140 (P-selection), respectively. The clotting time was examined by activated partially prothromplastin time (APTT) test. The fibrinogen adsorption on the drug-loaded PLGA films was evaluated by enzyme-linked immunosorbent assay (ELISA). All obtained data showed that incorporating curcumin in rapamycin-loaded PLGA coating can significantly decrease platelet adhesion and activation, prolong APTT clotting time as well as decrease the fibrinogen adsorption. All results indicated that incorporating curcumin in rapamycin-eluting coating obviously improve the blood compatibility of rapamycin-eluting stents. It was suggested that it may be possible to develop a drug-eluting stent which had the characteristics of not only good anti-proliferation but also improved anticoagulation.

Transcriptional regulation and the effects of sodium butyrate and glycosylation on catalytic activity of human germ cell alkaline phosphatase.

Human choriocarcinoma cells, the malignant trophoblasts, synthesize germ cell alkaline phosphatase (GCAP) which shares 98% sequence identity with the placental alkaline phosphatase (AP). The two isozymes are immunologically similar but react differentially toward inhibition by L-leucine or EDTA. Administration of sodium butyrate to choriocarcinoma cells greatly increased the transcription rate of the GCAP gene, resulting in an increase in mRNA expression and enzyme biosynthesis. The butyrate-modulated AP induction was blocked by cycloheximide, suggesting that a mediator protein may be involved. Protein sequence deduced from complementary DNA analysis suggests that GCAP contains two potential sites for asparagine (N)-linked glycosylation. The marked increase in GCAP expression by butyrate in choriocarcinoma cells allowed us to study the extent of N-linked glycosylation and its role on GCAP enzyme activity. After limited tunicamycin treatment, Mr 65,000 (fully processed), Mr 58,000 (nonglycosylated), and Mr 62,000 polypeptides were synthesized by these cells in the presence of butyrate. This suggests that the Mr 62,000 product may be the singly glycosylated GCAP monomer and that both sites are glycosylated in this phosphatase. The glycosylated and nonglycosylated GCAPs, synthesized by butyrate-treated choriocarcinoma cells in the absence or presence of tunicamycin, respectively, were similarly inhibited by L-leucine or EDTA. Moreover, the specific enzyme activity of glycosylated and nonglycosylated GCAP remained unchanged, indicating that AP lacking N-linked oligosaccharide side chains was catalytically active. This is supported by the finding that nonglycosylated GCAP incorporated inorganic phosphate which binds to the active site of AP. Since the active form of AP is a homodimer, our data indicate that the glycan moieties are not required for the dimerization and catalytic activity of GCAP.

Equatorial atmospheric Kelvin waves during El Niño episodes and their effect on stratospheric QBO.

Equatorial atmospheric Kelvin waves are investigated during a positive El Niño Southern Oscillation (ENSO) episode using temperature data retrieved from GPS Radio Occultation (RO) observations of FORMOSAT-3/COSMIC during the period from August 2006 to December 2013. Enhanced Kelvin wave amplitudes are observed during the El Niño episode of 2009-2010 and it is also observed that these amplitudes correlate with the Niño 3.4 index and also with outgoing longwave radiation and trade wind index. This study indicates that the enhanced equatorial atmospheric Kelvin wave amplitudes might be produced by geophysical processes that were involved in the onset and development of the El Niño episode. Further, easterly winds above the tropopause during this period favored the vertically upward propagation of these waves that induced a fast descending westerly regime by the end of 2010, where the zero-wind line is observed to take only 5 months to descend from 10 to 50 hPa. The current study presents observational evidence of enhanced Kelvin wave amplitudes during El Niño that has affected the stratospheric quasi-biennial oscillation (QBO) through wave-mean flow interactions. Earlier El Niño episodes of 1987 and 1998 are also qualitatively investigated, using reanalysis data. It is found that there might have been an enhancement in the equatorial Kelvin wave amplitudes during almost all El Niño episodes, however, an effect of a fast descending westerly is observed in the QBO only when the ambient zonal winds in the lower stratosphere favor the upward propagation of the Kelvin waves and consequently they interact with the mean flow. This study indicates that the El Niño and QBO are not linearly related and wave mean flow interactions play a very important role in connecting these two geophysical phenomena.

Immortalization of virus-free human placental cells that express tissue-specific functions.

Human pregnancy-specific glycoproteins (PSGs) are a family of closely related placental proteins that, together with the carcinoembryonic antigen members, comprise a subfamily within the immunoglobulin superfamily. To facilitate study of the control of PSG expression, we immortalized human placental cell lines with adenovirus-origin-minus (ori-)-simian virus-40 (SV40) recombinant viruses containing either wild-type or temperature-sensitive (ts) A mutants of SV40. Cells transformed with the SV40 tsA chimera (HP-A1 and HP-A2), but not the SV40 wild-type chimera (HP-W1), were temperature sensitive for transformation. All three cell lines expressed trophoblast-specific genes, including PSG and the alpha- and beta-subunits of hCG. Human CG alpha expression was greatly stimulated by (Bu)2cAMP in all three cell lines; shifting HP-A1 and HP-A2 cells to the nonpermissive temperature (39.5 C) further increased hCG alpha expression. At both 33 C (permissive temperature) and 39.5 C, the transformed placental cells expressed PSG mRNAs of 2.2 and 1.7 kilobases; expression was greatly stimulated by sodium butyrate. In the absence of an inducer, the three placental lines synthesized a PSG of 64 kilodaltons (kDa). In the presence of butyrate, they synthesized PSGs of 72, 64, and 54 kDa, similar to the placental PSGs. However, in placenta the predominant species is the 72-kDa product. At 39.5 C, butyrate selectively increased synthesis of the 72-kDa PSG in HP-A1 and HP-A2 cells. To characterize PSG promoter activity, we constructed chloramphenicol acetyltransferase (CAT) fusion genes containing -809 to -44 basepairs up-stream of the translational start site of the PSG6 gene. Using transient expression assays, we demonstrated that the -809/-44 region of the PSG6 gene contained cis-acting sequences that can direct CAT expression in human placental cells. Sodium butyrate, which stimulates PSG expression, greatly increased CAT activity, indicating that butyrate-induced PSG expression is regulated primarily at the level of gene transcription.

Inhibition of tyrosine aminotransferase gene expression by retinoic acid.

Regulation of tyrosine aminotransferase (TAT) gene expression was examined in RALA255-10G, a simian virus-40 tsA mutant-immortalized adult rat hepatocyte line. At the nonpermissive temperature (40 C), these hepatocytes exhibited a differentiated phenotype and actively expressed the TAT gene, but only in the presence of dexamethasone (DEX). The glucocorticoid-mediated TAT expression was inhibited by cycloheximide, a protein synthesis inhibitor, and by RU486, a glucocorticoid antagonist, suggesting that glucocorticoid induction requires protein synthesis and may be mediated through hormone receptors. (Bu)2cAMP (Bt2cAMP) or retinoic acid, individually or in combination, failed to increase TAT mRNA levels. However, Bt2cAMP greatly potentiated the induction by DEX, whereas retinoic acid inhibited the induction by DEX or DEX/Bt2cAMP. Nuclear run-on assays demonstrated that the induction of TAT expression by DEX or DEX/Bt2cAMP in RALA255-10G cells is regulated primarily at the transcriptional level. In contrast, retinoic acid antagonized the DEX- or DEX/Bt2cAMP-mediated induction without affecting the rate of TAT gene transcription. Instead, retinoic acid destabilized TAT mRNA. The half-life values of TAT mRNA in DEX/Bt2cAMP- and DEX/Bt2cAMP/retinoic acid-treated cells were approximately 235-270 min and 90-100 min, respectively. Our results indicate that inhibition of TAT expression by retinoic acid was regulated primarily at the posttranscriptional level.

[The ultrastructural and immunohistochemical observations of anaplastic meningioma].

Eighteen cases of anaplastic meningioma were studied by LM, EM and immunohistochemistry for vimentin, EMA, keratin, GFAP and S-100. Microscopically, there were four histologic types, i.e. fibrosarcoma-like, angiosarcoma-like, polymorphic giant cell sarcoma-like and angiopapillary structure. By EM, four kinds of cells: undifferentiated cell, intermediate transitional cell, spindle-shaped cell, and giant cell, were found and variant transitions from undifferentiated or poorly, differentiated to meningioma cells were observed. Their ultrastructures and immunohistochemical features are similar to those of malignant mesothelioma. Since these two kinds of neoplasm showed both mesenchymal and epithelial cells in the features, the authors consider that their histogenesis may also be similar.

Preparation and characterization of rapamycin-loaded PLGA coating stent.

In this study, using polylactic acid-co-glycolic acid (PLGA) with a molecular weight of 95,800 Da as drug carrier, three dose (low, moderate, high) rapamycin-eluting stents and the corresponding coating films were prepared. The pre- and post-expansion morphology of the rapamycin-eluting stent was examined by scanning electron microscopy (SEM), indicating that the coating was very smooth and uniform. The coating had the ability to withstand the compressive and tensile strains imparted without cracking from the stent during expansion process. There were many voids on stent coating surface after released for 18 days in release medium. The thermodynamics data of the stent coating film measured by differential scanning calorimetry (DSC) showed a lack of measurable solubility of rapamycin in the PLGA matrix. The release behavior of rapamycin from stent surface had a two phase release profile with a burst release period of about 2 days, followed by a sustained and slow release phase. The mass loss behavior of PLGA appeared linear throughout most of the degradation period, corresponding to an approximately constant mass loss rate. The platelet adhesion tests showed that the rapamycin-eluting films may have a good blood compatibility compared with control samples. Take into these results account, this novel rapamycin-eluting may be a good candidate to resolve in-stent restenosis.

Glucose-6-phosphate transporter gene therapy corrects metabolic and myeloid abnormalities in glycogen storage disease type Ib mice.

Glycogen storage disease type Ib (GSD-Ib) is caused by a deficiency in the glucose-6-phosphate transporter (G6PT), an endoplasmic reticulum-associated transmembrane protein that is ubiquitously expressed. GSD-Ib patients suffer from disturbed glucose homeostasis and myeloid dysfunctions. To evaluate the feasibility of gene replacement therapy for GSD-Ib, we have infused adenoviral (Ad) vector containing human G6PT (Ad-hG6PT) into G6PT-deficient (G6PT(-/-)) mice that manifest symptoms characteristics of the human disorder. Ad-hG6PT infusion restores significant levels of G6PT mRNA expression in the liver, bone marrow and spleen, and corrects metabolic as well as myeloid abnormalities in G6PT(-/-) mice. The G6PT(-/-) mice receiving gene therapy exhibit improved growth; normalized serum profiles for glucose, cholesterol, triglyceride, uric acid and lactic acid; and reduced hepatic glycogen deposition. The therapy also corrects neutropenia and lowers the elevated serum levels of granulocyte colony-stimulating factor. The development of bone and spleen in the infused G6PT(-/-) mice is improved and accompanied by increased cellularity and normalized myeloid progenitor cell frequencies in both tissues. This effective use of gene therapy to correct metabolic imbalances and myeloid dysfunctions in GSD-Ib mice holds promise for the future of gene therapy in humans.

Long-term correction of murine glycogen storage disease type Ia by recombinant adeno-associated virus-1-mediated gene transfer.

Glycogen storage disease type Ia (GSD-Ia) is caused by a deficiency in glucose-6-phosphatase-alpha (G6Pase-alpha), a nine-transmembrane domain, endoplasmic reticulum-associated protein expressed primarily in the liver and kidney. Previously, we showed that infusion of an adeno-associated virus (AAV) serotype 2 vector carrying murine G6Pase-alpha (AAV2-G6Pase-alpha) into neonatal GSD-Ia mice failed to sustain their life beyond weaning. We now show that neonatal infusion of GSD-Ia mice with an AAV serotype 1-G6Pase-alpha (AAV1-G6Pase-alpha) or AAV serotype 8-G6Pase-alpha (AAV8-G6Pase-alpha) results in hepatic expression of the G6Pase-alpha transgene and markedly improves the survival of the mice. However, only AAV1-G6Pase-alpha can achieve significant renal transgene expression. A more effective strategy, in which a neonatal AAV1-G6Pase-alpha infusion is followed by a second infusion at age one week, provides sustained expression of a complete, functional, G6Pase-alpha system in both the liver and kidney and corrects the metabolic abnormalities in GSD-Ia mice for the 57 week length of the study. This effective use of gene therapy to correct metabolic imbalances and disease progression in GSD-Ia mice holds promise for the future of gene therapy in humans.

In islet-specific glucose-6-phosphatase-related protein, the beta cell antigenic sequence that is targeted in diabetes is not responsible for the loss of phosphohydrolase activity.

AIMS/HYPOTHESIS: There are three members of the glucose-6-phosphatase (G6Pase) family: (1) the liver/kidney/intestine G6Pase-alpha (encoded by G6PC), which is a key enzyme in glucose homeostasis; (2) the ubiquitous G6Pase-beta (encoded by G6PC3); and (3) the islet-specific G6Pase-related protein (IGRP, encoded by /G6PC2). While G6Pase-alpha and G6Pase-beta are functional glucose-6-phosphate hydrolases, IGRP possesses almost no hydrolase activity. This was unexpected since G6Pase-alpha is more closely related to IGRP than G6Pase-beta. Recently, amino acids 206-214 in IGRP were identified as a beta cell antigen targeted by a prevalent population of pathogenic CD8+ T cells in autoimmune diabetes, suggesting that this peptide confers functional specificity to IGRP. We therefore investigated the molecular events that inactivate IGRP activity and the effects of the beta cell antigen sequence on the stability and enzymatic activity of G6Pase-alpha. METHODS: Studies were performed using site-directed mutagenesis and transient expression assays. Protein stability was evaluated by Western blotting, proteasome inhibitor studies and in vitro transcription-translation. RESULTS: We showed that the residues responsible for G6Pase activity are more extensive than previously recognised. Introducing the IGRP antigenic motif into G6Pase-alpha does not completely destroy activity, although it does destabilise the protein. The low hydrolytic activity in IGRP is due to the combination of multiple independent mutations. CONCLUSIONS/INTERPRETATION: The loss of catalytic activity in IGRP arises from the sum of many sequence differences. G6Pase-alpha mutants containing the beta cell antigen sequence are preferentially degraded in cells, which prevents targeting by pathogenic CD8+ T cells. It is possible that IGRP levels in beta cells could dictate susceptibilities to diabetes.

Human variant glucose-6-phosphate transporter is active in microsomal transport.

Glycogen storage disease type lb (GSD-lb) is caused by deficiencies in the glucose-6-phosphate transporter (G6PT), which works together with glucose-6-phosphatase to maintain glucose homeostasis. In humans, there are two alternatively spliced transcripts, G6PT and variant G6PT (vG6PT), differing by the inclusion of a 66-bp exon-7 sequence in vG6PT. We have previously shown that the G6PT protein functions as a microsomal glucose-6-phosphate (G6P) transporter, which is anchored to the endoplasmic reticulum by ten transmembrane helices. Here, we demonstrate that vG6PT is also active in microsomal G6P transport. The additional 22 amino acids in vG6PT is predicted to constitute a part of the luminal loop 4. Our data indicate that this loop plays no vital role in microsomal G6P transport. Further, we show that G6PT mRNA is expressed in all organs and tissues examined, but that the vG6PT transcript is expressed exclusively in the brain, heart, and skeletal muscle. These results raise the possibility that mutations in exon-7 of the G6PT gene, which would not perturb glucose homeostasis, might have other deleterious effects.