A new spectrin, beta IV, has a major truncated isoform that associates with promyelocytic leukemia protein nuclear bodies and the nuclear matrix.

We isolated cDNAs that encode a 77-kDa peptide similar to repeats 10-16 of beta-spectrins. Its gene localizes to human chromosome 19q13.13-q13.2 and mouse chromosome 7, at 7.5 centimorgans. A 289-kDa isoform, similar to full-length beta-spectrins, was partially assembled from sequences in the human genomic DNA data base and completely cloned and sequenced. RNA transcripts are seen predominantly in the brain, and Western analysis shows a major peptide that migrates as a 72-kDa band. This new gene, spectrin betaIV, thus encodes a full-length minor isoform (SpbetaIVSigma1) and a truncated major isoform (SpbetaIVSigma5). Immunostaining of cells shows a micropunctate pattern in the cytoplasm and nucleus. In mesenchymal stem cells, the staining concentrates at nuclear dots that stain positively for the promyelocytic leukemia protein (PML). Expression of SpbetaIVSigma5 fused to green fluorescence protein in cells produces nuclear dots that include all PML bodies, which double in number in transfected cells. Deletion analysis shows that partial repeats 10 and 16 of SpbetaIVSigma5 are necessary for nuclear dot formation. Immunostaining of whole-mount nuclear matrices reveals diffuse positivity with accentuation at PML bodies. Spectrin betaIV is the first beta-spectrin associated with a subnuclear structure and may be part of a nuclear scaffold to which gene regulatory machinery binds.

Downeast anemia (dea), a new mouse model of severe nonspherocytic hemolytic anemia caused by hexokinase (HK(1)) deficiency.

A new spontaneous mutation in the A/J inbred mouse strain, downeast anemia (dea), causes severe hemolytic anemia with extensive tissue iron deposition and marked reticulocytosis. The anemia is present at birth and persists throughout life. The defect is inherited as an autosomal recessive and is transferable through bone marrow stem cells. The red cell morphology is consistent with a nonspherocytic hemolytic anemia, suggestive of a red cell enzymopathy. In linkage analysis, dea is nonrecombinant with the hexokinase-1 gene (Hk1) on mouse Chromosome 10. Expression of Hk1 is markedly decreased in dea erythroid tissues, and the transcript produced is larger than normal. Hexokinase enzyme activity is significantly decreased in dea tissues, including red cells, spleen, and kidney. Southern blot analyses revealed approximately 5.5 kb of additional sequence in the 5' portion of the dea Hk1 gene, which was identified by direct sequencing as an early transposon (ETn) insertion in intron 4. ETn insertions disrupt genes in several mouse models by a variety of mechanisms, including aberrant splicing of ETn sequences into the mRNA. We conclude that the primary gene defect in the dea mutation is in Hk1 and that dea is a model of generalized hexokinase deficiency, the first such model identified to date.

Defects in the cappuccino (cno) gene on mouse chromosome 5 and human 4p cause Hermansky-Pudlak syndrome by an AP-3-independent mechanism.

Defects in a triad of organelles (melanosomes, platelet granules, and lysosomes) result in albinism, prolonged bleeding, and lysosome abnormalities in Hermansky-Pudlak syndrome (HPS). Defects in HPS1, a protein of unknown function, and in components of the AP-3 complex cause some, but not all, cases of HPS in humans. There have been 15 inherited models of HPS described in the mouse, underscoring its marked genetic heterogeneity. Here we characterize a new spontaneous mutation in the mouse, cappuccino (cno), that maps to mouse chromosome 5 in a region conserved with human 4p15-p16. Melanosomes of cno/cno mice are immature and dramatically decreased in number in the eye and skin, resulting in severe oculocutaneous albinism. Platelet dense body contents (adenosine triphosphate, serotonin) are markedly deficient, leading to defective aggregation and prolonged bleeding. Lysosomal enzyme concentrations are significantly elevated in the kidney and liver. Genetic, immunofluorescence microscopy, and lysosomal protein trafficking studies indicate that the AP-3 complex is intact in cno/cno mice. It was concluded that the cappuccino gene encodes a product involved in an AP-3-independent mechanism critical to the biogenesis of lysosome-related organelles. (Blood. 2000;96:4227-4235)

Targeted disruption of the beta adducin gene (Add2) causes red blood cell spherocytosis in mice.

Adducins are a family of cytoskeleton proteins encoded by three genes (alpha, beta, gamma). In a comprehensive assay of gene expression, we show the ubiquitous expression of alpha- and gamma-adducins in contrast to the restricted expression of beta-adducin. beta-adducin is expressed at high levels in brain and hematopoietic tissues (bone marrow in humans, spleen in mice). To elucidate adducin's role in vivo, we created beta-adducin null mice by gene targeting, deleting exons 9-13. A 55-kDa chimeric polypeptide is produced from the first eight exons of beta-adducin and part of the neo cassette in spleen but is not detected in peripheral RBCs or brain. beta-adducin null RBCs are osmotically fragile, spherocytic, and dehydrated compared with the wild type, resembling RBCs from patients with hereditary spherocytosis. The lack of beta-adducin in RBCs leads to decreased membrane incorporation of alpha-adducin (30% of normal) and unexpectedly promotes a 5-fold increase in gamma-adducin incorporation into the RBC membrane skeleton. This study demonstrates adducin's importance to RBC membrane stability in vivo.

Mild spherocytosis and altered red cell ion transport in protein 4. 2-null mice.

Protein 4.2 is a major component of the red blood cell (RBC) membrane skeleton. We used targeted mutagenesis in embryonic stem (ES) cells to elucidate protein 4.2 functions in vivo. Protein 4. 2-null (4.2(-/-)) mice have mild hereditary spherocytosis (HS). Scanning electron microscopy and ektacytometry confirm loss of membrane surface in 4.2(-/-) RBCs. The membrane skeleton architecture is intact, and the spectrin and ankyrin content of 4. 2(-/-) RBCs are normal. Band 3 and band 3-mediated anion transport are decreased. Protein 4.2(-/-) RBCs show altered cation content (increased K+/decreased Na+)resulting in dehydration. The passive Na+ permeability and the activities of the Na-K-2Cl and K-Cl cotransporters, the Na/H exchanger, and the Gardos channel in 4. 2(-/-) RBCs are significantly increased. Protein 4.2(-/-) RBCs demonstrate an abnormal regulation of cation transport by cell volume. Cell shrinkage induces a greater activation of Na/H exchange and Na-K-2Cl cotransport in 4.2(-/-) RBCs compared with controls. The increased passive Na+ permeability of 4.2(-/-) RBCs is also dependent on cell shrinkage. We conclude that protein 4.2 is important in the maintenance of normal surface area in RBCs and for normal RBC cation transport.

Intracisternal A-particle element transposition into the murine beta-glucuronidase gene correlates with loss of enzyme activity: a new model for beta-glucuronidase deficiency in the C3H mouse.

The severity of human mucopolysaccharidosis type VII (MPS VII), or Sly syndrome, depends on the relative activity of the enzyme beta-glucuronidase. Loss of beta-glucuronidase activity can cause hydrops fetalis, with in utero or postnatal death of the patient. In this report, we show that beta-glucuronidase activity is not detectable by a standard fluorometric assay in C3H/HeOuJ (C3H) mice homozygous for a new mutation, gusmps2J. These gusmps2J/gusmps2J mice are born and survive much longer than the previously characterized beta-glucuronidase-null B6.C-H-2(bm1)/ByBir-gusmps (gusmps/gusmps) mice. Northern blot analysis of liver from gusmps2J/gusmps2J mice demonstrates a 750-bp reduction in size of beta-glucuronidase mRNA. A 5.4-kb insertion in the Gus-sh nucleotide sequence from these mice was localized by Southern blot analysis to intron 8. The ends of the inserted sequences were cloned by inverse PCR and revealed an intracisternal A-particle (IAP) element inserted near the 3' end of the intron. The sequence of the long terminal repeat (LTR) regions of the IAP most closely matches that of a composite LTR found in transposed IAPs previously identified in the C3H strain. The inserted IAP may contribute to diminished beta-glucuronidase activity either by interfering with transcription or by destabilizing the message. The resulting phenotype is much less severe than that previously described in the gusmps/gusmps mouse and provides an opportunity to study MPS VII on a genetic background that clearly modulates disease severity.

The gene encoding protein 4.2 is distinct from the mouse platelet storage pool deficiency mutation pallid.

Previous studies identified the gene encoding the erythrocyte membrane protein 4.2 (Epb4.2) as a candidate for the mouse mutation pallid (pa); Epb4.2 genetically colocalized near pa on mouse Chromosome 2, and a truncated Epb4.2 transcript was present in tissues derived from pallid mice. We report here evidence that Epb4.2 and pa are not allelic. The pallid cDNA and intron/exon boundaries show no significant variation from the known BALB/c and C57BL/6J Epb4.2 sequence, and normal immunoreactive 72-kDa protein 4.2 is present in pallid tissues. Two recombinations between Epb4.2 and pa were identified in 173 phenotypically mutant (C57BL/6J-pa/pa x Mus castaneus) F2 animals. Northern blotting reveals a truncated Epb4.2 transcript in kidney mRNA from normal wild Mus domesticus (WSB/Ei) mice that comigrates with the pallid Epb4.2 mRNA. As the pa mutation originally arose in a wild M. domesticus mouse, we conclude that the Epb4.2 mRNA characteristic of pallid is a normal polymorphism derived from its wild ancestor and that Epb4.2 and pa are distinct loci.

Murine mucopolysaccharidosis type VII: long term therapeutic effects of enzyme replacement and enzyme replacement followed by bone marrow transplantation.

We demonstrated previously that short term administration of recombinant beta-glucuronidase to newborn mice with mucopolysaccharidosis type VII reduced lysosomal storage in many tissues. Lysosomal storage accumulated gradually after cessation of enzyme replacement therapy. Mice alive at 1 yr of age had decreased bone deformities and less lysosomal storage in cortical neurons. Here we compare the effects of long term enzyme replacement initiated either at birth or at 6 wk of age, and of enzyme administration initiated at birth followed by syngeneic bone marrow transplantation (BMT) at 5 wk of age. Several mice from each treatment group lived to at least 1 yr of age. Liver and spleen samples had beta-glucuronidase levels ranging from 2.4 to 19.8% of normal and showed a parallel decrease in lysosomal storage. The combination of enzyme replacement therapy followed by BMT reduced lysosomal distension in meninges, corneal fibroblasts, and bone when compared with treatment with enzyme alone. Mice treated at birth had less lysosomal storage in some neurons of the brain and the skeletal dysplasia was less severe when compared to mice whose treatment was delayed until 6 wk of age. We conclude that both enzyme replacement alone and early enzyme replacement followed by BMT have long term positive effects on murine mucopolysaccharidosis type VII. In addition, treatment started at birth is far more effective than treatment initiated in young adults.

Anion exchanger 1 (band 3) is required to prevent erythrocyte membrane surface loss but not to form the membrane skeleton.

The red blood cell (RBC) membrane protein AE1 provides high affinity binding sites for the membrane skeleton, a structure critical to RBC integrity. AE1 biosynthesis is postulated to be required for terminal erythropoiesis and membrane skeleton assembly. We used targeted mutagenesis to assess AE1 function in vivo. RBCs lacking AE1 spontaneously shed membrane vesicles and tubules, leading to severe spherocytosis and hemolysis, but the levels of the major skeleton components, the synthesis of spectrin in mutant erythroblasts, and skeletal architecture are normal or nearly normal. The results indicate that AE1 does not regulate RBC membrane skeleton assembly in vivo but is essential for membrane stability. We postulate that stabilization is achieved through AE1-lipid interactions and that loss of these interactions is a key pathogenic event in hereditary spherocytosis.

Genetic localization of Cd63, a member of the transmembrane 4 superfamily, reveals two distinct loci in the mouse genome.

The membrane protein CD63, a molecular marker for early stages of melanoma progression, has been associated with platelet storage pool deficiency disorders (SPD). CD63 localizes to the membranes of platelets, lysosomes, and melanosomes, all of which are affected in a specific subgroup of SPD. The cDNA encoding CD63 detects two closely related sequences that map to different regions of the mouse genome. One locus maps to mouse Chromosome (Chr) 10 in a region that shares linkage homology with the human chromosome encoding human CD63. The second locus maps to mouse Chr 18 in a region that bears no known human CD63-related genes. No SPD has been localized to these regions of either the mouse or the human chromosomes.

Direct access surgery.

Evolving surgical practice in the UK is imposing increasing pressures on surgical outpatient clinics. A Direct Access Surgery (DAS) programme was evaluated in which otherwise healthy patients with simple surgical conditions presenting little diagnostic difficulty were referred directly to hospital for surgical operation without attending the outpatient department. Specific referral criteria for DAS were devised. Appropriate surgical conditions or procedures included hernias, vasectomies, cystic scrotal swellings, subcutaneous lumps and skin lesions, symptomatic gallstones, varicose veins and ingrowing toenails. Over a 12 month period, 105 patients were referred for DAS and 102 underwent operation. There was a low incidence of inaccurate diagnosis (< 2%) and inappropriate referral (< 1%). DAS was well received by both General Practitioners (GPs) and patients. Implementation of such programmes may result in considerable savings of outpatient time and resources.

Falsely elevated ankle pressures in severe leg ischaemia: the pole test--an alternative approach.

Ankle-branchial pressure indices (ABPIs), measured by sphygmomanometer and Doppler probe, are an accepted index of chronic leg ischaemia. However, tibial artery sclerosis or calcification decreases compliance, producing falsely elevated cuff occlusion pressures. Arterial cannulation is invasive and impractical, but by elevating the foot and measuring the height at which the Doppler signal disappears, ankle systolic pressure in mmHg can be derived. Using an 8MHz Doppler apparatus and calibrated pole, ankle systolic pressures measured by sphygmomanometer and elevation were compared in 49 severely ischaemic legs (40 patients). ABPIs were derived by dividing ankle systolic pressure by brachial pressure. Median (interquartile range) ABPI assessed by sphygmomanometry was 0.46 (0.35-0.56). Median ABPI measured by leg elevation was significantly lower at 0.21 (0.14-0.30), p < 0.0001, Wilcoxon. In 20 patients undergoing in situ vein bypass grafting, direct transducer-derived pressure measurements were obtained. Median ABPI for this method was 0.15 (0.11-0.27). No significant difference was found when compared with ABPIs derived by elevation, median 0.2 (0.13-0.31), p = 0.324, however median ABPI measured by sphygmomanometry was significantly higher at 0.37 (0.27-0.6), p = 0.0008. Correlation of elevation with transducer-assessed pressure measurements (r = 0.88) was closer than with cuff-derived measurements (r = 0.69). Pressures derived by leg elevation provide a more accurate index of severe leg ischaemia than sphygmomanometry, although the technique is limited to assessing pressures of less than approximately 60 mmHg. Falsely elevated ABPIs may underestimate the extent of disease in patients assessed for vascular reconstruction.

Treatment of murine mucopolysaccharidosis type VII by syngeneic bone marrow transplantation in neonates.

BACKGROUND: Bone marrow transplantation (BMT) proved an effective therapy for murine mucopolysaccharidosis type VII (MPS VII) in adult gusmps/gusmps mice with well developed clinical and pathologic characteristics of the disease. MPS VII mice transplanted as adults had a marked decrease in lysosomal storage material in many organs, although not in the skeleton and brain (1). Since untreated newborn MPS VII mice appear normal and have minimal lysosomal storage material detectable microscopically, we postulated that BMT in newborn mice might prevent the subsequent accumulation of storage material. EXPERIMENTAL DESIGN: One-day-old mutant and phenotypically normal mice were exposed to 2, 4, 6 and 8 Gray and then injected intravenously with syngeneic bone marrow cells from homozygous normal females. Transplanted mice were examined biochemically and microscopically at 10 weeks and 10 months of age. RESULTS: Newborn mice receiving BMT lived longer than untreated mutants, had less severe facial dysmorphism, and better mobility. beta-Glucuronidase activity in liver, spleen, kidney and brain increased with increasing radiation dose. The secondary elevations of alpha-galactosidase and beta-hexosaminidase observed in MPS VII, were significantly reduced in liver and spleen in all radiation groups. Treated mutants had less histologic evidence of lysosomal storage disease in bones, joints and periarticular tissue as compared with untreated mutants. Neonatal BMT also reduced storage in the leptomeninges, ependyma and retinal pigment epithelium and caused a slight decrease in neuronal storage at high radiation dose. Radiation dose dependent cerebellar and retinal dysplasia and long bone growth retardation was observed when the therapy was initiated in newborn mice but not when the animals were transplanted as adults. CONCLUSIONS: BMT is a more effective therapy for MPS VII when it is performed at birth rather than in adults. Alternate means of ablating host hematopoietic stem cells should be employed as a pretreatment for BMT due to the severe side effects of radiation on newborns.

Reversal of pathology in murine mucopolysaccharidosis type VII by somatic cell gene transfer.

An inherited deficiency of beta-glucuronidase in humans, mice and dogs causes mucopolysaccharidosis VII (Sly syndrome), a progressive degenerative disease that reduces lifespan (to an average of 5 months in mice) and results from lysosomal storage of undegraded glycosaminoglycans in the spleen, liver, kidney, cornea, brain and skeletal system. Bone marrow transplantation in mutant mice provides a source of normal enzyme ('cross-correction'), which substantially improves the clinical condition and extends the average lifespan to 18 months. Gene therapy by transfer of a beta-glucuronidase gene into mutant haematopoietic stem cells is an alternative approach, but it is not known whether the low expression of vector-transferred genes in vivo would be sufficiently effective. Here we show that retroviral vector-mediated transfer of the gene to mutant stem cells results in long-term expression of low levels of beta-glucuronidase which partially corrects the disease by reducing lysosomal storage in liver and spleen.

Tissue- and cell-specific expression of human sn-glycerol-3-phosphate dehydrogenase in transgenic mice.

Comparison of the promoter sequence for the sn-glycerol-3-phosphate dehydrogenase (GPDH, EC 1.1.1.8) genes in mice and humans showed that there were three promoter domains conserved in evolution (1). To study the functional organization of the GPDH promoter, we generated transgenic mice carrying the complete human gene, GPD1. The level of human and mouse GPDH activity was measured in each tissue and the amount of human-mouse GPDH heterodimer was used as a sensitive indicator of cell-specific expression of GPD1. During postnatal development and in adult tissues of the transgenic mice, human GPDH was expressed at levels that corresponded closely to the expression of the endogenous mouse gene, Gdc-1. Surprisingly, deletion of the evolutionarily conserved fat-specific elements (FSE) in the proximal promoter region failed to reveal any alterations in GPD1 expression that were specific for either white or brown adipose tissue.

Increased life span and correction of metabolic defects in murine mucopolysaccharidosis type VII after syngeneic bone marrow transplantation.

The gusmps/gusmps mouse has no beta-glucuronidase activity and develops murine mucopolysaccharidosis type VII (MPS VII). The clinical and pathologic abnormalities are similar to those found in humans with severe MPS VII. Mutant mice are dysmorphic, dwarfed, and have a shortened life span. Pathologic findings include widespread lysosomal storage. To determine whether bone marrow transplantation (BMT) corrects these abnormalities, genetically identical mutant animals were given syngeneic bone marrow transplants using cells from +/+ mice. Initial experiments showed that levels of beta-glucuronidase activity in recipient tissues correlated with the amount of radiation administered before BMT. Two groups of mice given BMT therapy were observed for periods of 1 and 2 years, respectively. These mice were evaluated using a combination of clinical, biochemical, histochemical, and pathologic analyses. Spleen, liver, cornea, and glomerular mesangial cells showed essentially complete correction at all radiation doses. Storage was partially corrected in meninges and perivascular cells in brain, and in renal tubular epithelial cells at the higher radiation doses. Life span in BMT-treated animals was increased approximately three-fold, approaching that seen in normal mice after BMT. These results support the position that BMT has a place in the therapeutic regimen for MPS VII.

Sequence conservation and structural organization of the glycerol-3-phosphate dehydrogenase promoter in mice and humans.

Cloned segments of the mouse glycerol-3-phosphate dehydrogenase (GPDH) gene, Gdc-1, were used to screen a human library. Human clones obtained spanned 25 kilobases of genomic DNA containing the human GPDH gene, GPD1. The 4 kb of sequence obtained from the 5'-flanking region and first exon of GPD1 was compared with the corresponding mouse sequence. Both sequences share a HindIII site located in what has proven to be the highly conserved 3' untranslated region of an upstream gene of unknown function, D15Kzl. The 3.6-kilobase segment of mouse DNA located between D15Kzl and Gdc-1 was provisionally termed the GPDH promoter. Alignment of the mouse promoter with the corresponding human sequence revealed two conserved domains. An upstream distal promoter region is approximately 900 base pairs in length. A downstream or proximal promoter region consists of approximately 300 base pairs immediately upstream of a TATA-like box and contains the fat-specific elements 1 and 2. Analysis of the chromatin structure of the Gdc-1 promoter revealed four DNase I-hypersensitive sites. They were present in DNA of liver and brown fat, in which GPDH expression is high, but were absent in DNA of spleen, in which GPDH expression is low. Methylation studies of the promoter showed it to be heavily methylated in sperm. However, the DNA from each adult somatic tissue had a unique distribution of nonmethylated sites and could easily be identified by its methylation pattern. These data suggest a structural model of the promoter that explains how Gdc-1 expression is differentially regulated in many types of cells.