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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Diabetes Center, Albert Einstein College of
Medicine, Bronx, New York; Department of Molecular and Cellular
Biology, Roswell Park Cancer Institute, Buffalo, New York; Cell Biology
and Metabolism Branch at the National Institute of Child Health and
Human Development, National Institutes of Health, Bethesda, Maryland;
and Department of Biochemistry, Faculty of Life Sciences, Imperial
College, London, United Kingdom.
Hermansky-Pudlak syndrome (HPS) is an inherited hemorrhagic disease
affecting the related subcellular organelles platelet dense granules,
lysosomes, and melanosomes. The mouse genes for HPS, pale ear and
pearl, orthologous to the human HPS1 and HPS2 (ADTB3A)
genes, encode a novel protein of unknown function and the
Hermansky-Pudlak syndrome (HPS) is an inherited
human disease affecting several intracellular organelles including
melanosomes, platelet dense granules, and lysosomes.1-3
Abnormalities in these 3 organelles cause hypopigmentation, prolonged
bleeding times, and in some patients ceroid deposition in several
tissues including lung. Associated clinical problems include severe
visual deficiencies, hemorrhaging requiring repeated platelet
transfusions, and fibrotic lung disease often leading to premature
death in midlife. Only symptomatic treatment is available.
The syndrome has been described in animal models such as the mouse
where at least 15 genetically distinct mutants occur.4,5 The disease is likewise genetically heterogeneous in humans where 3 distinct forms of HPS (HPS1, HPS2, and HPS3) have been
described.1,2,6,7 HPS1 comprises the majority of HPS
patients and is caused by a mutation in a chromosome 10 gene,
which produces a novel 79-kd protein product.8 This
protein is found predominantly in a cytosolic complex; its function
remains unknown.9,10 Mutations in the The corresponding or orthologous mouse models for HPS1 and HPS2 are the
pale ear11,12 and pearl13-15 pigment mutants,
respectively. The phenotypes of the 2 mouse mutants share significant
features in hypopigmentation, lysosomal secretion abnormalities, and
platelet physiology, possibly consistent with action on a common
organelle regulatory pathway. On the other hand, the mutant mice differ somewhat in pigmentation, lysosomal secretion in certain cell types,
and in quantity of the contents of platelet dense
granules.4 Although it is certain that the HPS2
(ADTB3A) gene affects vesicle trafficking, and it is
highly likely from phenotypes of pale ear mice and patients with HPS1
that this gene does likewise, it is less certain whether the 2 genes
act independently or by common pathways. This is especially true in
regard to whole animal phenotypes and patient clinical characteristics.
The fact that each mouse model is maintained as a congenic mutant on
the C57BL/6J background enables genetically controlled in vivo tests
for interaction of these 2 important genes. A classical method of
testing for interaction of genes in mutant mice is to analyze double
mutant offspring that contain mutant versions of both
genes.16
The pale ear and pearl genes are important not only because of their
critical roles in organelle production, function, and trafficking but
also because they are the corresponding mouse models for 2 forms of HPS
thus far identified in humans, including the most common form, HPS1.
HPS1 is the most common form as a result of a founder mutation in
Puerto Rico. We report that the HPS1 and ADTB3A
genes cooperate in independent pathways in the formation/function
of platelet dense granules, melanosomes, and lysosomes at the level of
the whole organism. Further, these double mutant mice are
experimentally useful in the study of HPS because they exhibit a
particularly severe form of the disease.
Mice
Genotyping
PCR primers (23F9: 5'-GAAATGGGGCTGCACATAG-3' and 17R3: 5'-GAACCCTCACACAGGACTCG-3') that specifically amplify the genomic junction point (678-bp product) in the pe genomic duplication17 were used to detect the pearl allele. However, this test cannot differentiate heterozygous pe/+ from homozygous pe/pe mice. Therefore, microsatellite markers D13Mit145 and D13Mit159, which are 0.3 cM distal and 0.5 cM proximal, respectively, to the pe locus,13 were used to type polymorphisms between C3H and C57BL/6J backgrounds to confirm the homozygosity of the pearl mutation allele (pe) in double mutants. Primers for amplification of microsatellite markers D13Mit145 and D13Mit159 were purchased from Research Genetics (Huntsville, AL). The PCR products of microsatellite markers were separated by 8% polyacrylamide gel electrophoresis and visualized with ethidium bromide. Other PCR products were separated by 1% agarose gel electrophoresis and visualized with ethidium bromide. Bleeding times and platelet collection Tail bleeding times were determined as described.18 Platelets were harvested from the peripheral blood of normal and mutant mice in the presence of sodium citrate.18Platelet serotonin assays Platelets were lysed in 1 mL distilled water and assayed fluorometrically for serotonin.18Mepacrine uptake Platelets were incubated with mepacrine and analyzed with a Leitz MPV-2 fluorescent microscope.19Thrombin-stimulated platelet secretion Platelets at 109 cells/mL were washed 2 times in phosphate-buffered saline containing 2% bovine serum albumin (BSA). For measurements of secretion of lysosomal enzymes and -granule
contents, platelets were treated with 1.6 U/mL thrombin (Sigma
Chemical, St Louis, MO) for 3 minutes with constant
shaking.20 The reaction was stopped with 2.5 nmol/mL of
Thromstop (American Diagnostics, Greenwich, CT).
Urine and tissue collection To amplify lysosomal enzyme concentrations in kidney and urine, female mice were treated for 20 days with testosterone. Testosterone greatly amplifies both the synthesis and secretion of lysosomal enzymes in kidney proximal tubule cells.21 At days 20 to 24, mice were placed in metabolism cages at 2 to 4 per cage and urine was collected at 24-hour intervals. After day 24, mice were killed by anoxia with CO2 and tissues were homogenized and stored frozen.Enzyme assays  -Glucuronidase and -galactosidase were assayed with
fluorescent methylumbelliferyl substrates.18 Protein was
determined with the Bio-Rad protein assay system (Bio-Rad
Laboratories, Hercules, CA).
Immunoblotting Fibrinogen and platelet factor 4 (PF4) were measured in platelets and platelet secretions by immunoblotting using rabbit antiserum to human fibrinogen (Diagnostica Stago, Asnieres, France) and a rabbit polyclonal antibody to rat PF4.22 All blots were exposed to film for several different lengths of time to ensure that the density of bands was within the linear range. Equivalent loading and transfer were verified by India ink staining of blots.Electron microscopy Eyes from 2 animals per each genetic group were immersion fixed with 4% formaldehyde, 2% glutaraldehyde, and 3% sucrose in 100 mM Hepes buffer at pH 7.4 for 10 minutes. Eyes were then cut, exposing the internal surface of the retina to the fixative. After fixation, portions of the eye, including the retina and the choroid, were further dissected to longitudinal strips posterior to the ora serrata and anterior to the macula. Tissues were rinsed in buffer and treated with 2% reduced osmium tetroxide for 1 hour. Tissues were rinsed then dehydrated in increasing graded series of ethanol and embedded in Eponate 12 (Ted Pella, Redding, CA) as in standard processing techniques for transmission electron microscopy. Cross-sections through the retina and choroid were obtained using a diamond knife and were contrasted with solutions of 3% uranyl acetate and lead citrate. Sections were viewed and photographed with a Philips CM-10 transmission electron microscope (Philips Electron Optics, FEI, Hillsboro, Oregon).Platelets were harvested from the peripheral blood of C57BL/6J, pale ear, pearl, and pale ear/pearl double mutant mice in the presence of sodium citrate. Platelets were pelleted gently and fixed in 2% paraformaldehyde/1.5% glutaraldehyde. The pellets were osmicated, stained with tannic acid and embedded in Epon for ultrathin section electron microscopy.
Breeding and pigmentation phenotypes of double mutant mice As the first step in producing double mutant mice, homozygous pale ear and homozygous pearl mice were mated to obtain F1 (ep/+, pe/+) offspring. These F1 animals, of normal black coat and eye color, were intercrossed, and F2 animals were typed as homozygous pearl (pe/pe), pale ear (ep/ep), or double mutants (ep/ep, pe/pe) by both coat color and molecular genotyping of the ep or pe mutations. Double mutant pale ear/pearl mice appear robust for at least 5 months of age though breeding performance is somewhat depressed.The majority of resultant F2 progeny had black coat and eye
colors identical to C57BL/6J normal mice. Some exhibited the same eye
(ie, dilute red in newborn and black in adults) and coat colors as
either pale ear or pearl mice. Adult pale ear mice display hypopigmentation of the ears and tails with no effects on pigmentation of other body parts (Figure 1). The pearl
mutant shows a brown-gray coat color on all body parts. In contrast, a
third group of offspring displayed a new phenotype (Figure 1) with
strikingly lighter coat color throughout than either parental single
mutant and light red eye color in both newborn and adult animals. These
animals were tentatively designated as double mutants (ep/ep,
pe/pe), a classification subsequently confirmed by PCR genotyping
of genomic DNA (not shown). By visual examination, the ep/+,
pe/pe mouse is not different from the +/+, pe/pe mouse
in coat color; neither is the ep/ep, pe/+ mouse different
from the ep/ep, +/+ mouse (not shown).
Double mutants or those of genotypes ep/+, pe/pe or ep/ep, pe/+ were selected for further breeding. As expected from mendelian genetics, self-matings of the double mutants produced the identical distinctive coat and eye color phenotype of the parents in all subsequent generations. Also, when double mutants were backcrossed to either pale ear or pearl mice, only pale ear or pearl phenotypes, respectively, were observed. Ultrastructure of eye melanosomes Eye tissues of control C57Bl/6J, pale ear, pearl, and double mutant mice were subjected to ultrastructural analysis by electron microscopy. Both quantitative and qualitative morphologic abnormalities in melanosomes of the retinal pigment epithelium (RPE) and choroid were apparent (Figure 2).
In the pearl mouse (Figure 2B), very small numbers of melanosomes were observed in the RPE. Melanosomes were present in the choroid of pearl mice, but they were reduced in number and exhibited greater size heterogeneity as compared with control tissues. Melanized melanosomes were extremely rare in the RPE cells of the pale ear mouse (Figure 2C). Fewer but abnormally large melanosomes (macromelanosomes) were observed in the pale ear choroid relative to wild-type. Less pigmented intermediate structures were also observed. In the double mutant mouse (Figure 2D), essentially no melanized melanosomes were observed in RPE cells, a situation similar to that of the pale ear RPE but different from that of pearl. Most striking, and in dramatic difference to that observed in either single mutant, was a drastic decrease in number of choroidal melanosomes in the double mutant. Only a very few multivesicular pigment granules with irregular boundaries and containing aggregates of melanosomes of various developmental stages were observed. Platelet characteristics As expected, double mutant mice had prolonged bleeding times. Four double mutants had bleeding times more than 15 minutes, a value at least as great as those observed in single mutant pale ear23 or pearl24 mice. It is technically difficult to determine if mutant mice have bleeding times longer than 15 minutes. Therefore, other platelet granule characteristics were assayed to determine if the presence of both mutant genes caused more severe effects. Platelet serotonin levels are an accurate monitor of the contents of platelet dense granules, which supply several low-molecular-weight compounds critical for normal hemostasis. As previously documented,23,24 platelet serotonin levels are significantly depressed in both the pale ear and pearl mutants with pale ear having intermediate levels (Table 1). More notably, a particularly severe depression occurs in double mutant platelets to a level only 1% that of C57BL/6J controls. All mutant platelet serotonins are significantly different (P < .001), not only from C57BL/6J, but from all other mutant values.
Most mouse HPS mutants have abnormalities in secretion of the contents
of platelet lysosomes, and similar abnormalities have been observed in
patients with HPS.4 Previous studies23,24 had
documented relatively high thrombin-stimulated secretion of lysosomal
enzymes from platelets of pale ear and low secretion from platelets of
pearl. The present analyses (Table 2) of
the release of lysosomal enzymes from platelets of ep/ep, pe/pe
(double mutant) mice revealed a phenotype similar to that of
pearl, that is, low rates of secretion, for each of 2 lysosomal
enzymes,
Contents of the major platelet subcellular organelle, the
Ultrastructural analyses of platelets by electron microscopy (Figure
4) were consistent with the above
findings. Very few, if any, dense granules with their typical
"bull's eye" appearance25 were found in the double
mutant mouse platelets (Figure 4D), although these structures were
readily apparent in normal mice (Figure 4A) and occasionally observed
in pale ear mutants (Figure 4B). Further, consistent with the above
immunoblotting analyses of
Tissue steady-state lysosomal enzyme levels and secretion of kidney lysosomal enzymes Measurements of tissue steady-state levels of lysosomal enzymes revealed significant and potentially important differences between the single and double mutants (Table 3). Murine kidney undergoes significant hypertrophy, increases in lysosomal enzyme levels in proximal tubule cells and hypersecretion of lysosomal enzymes from these cells into urine in response to testosterone treatment.21 As previously documented,26 both pale ear and pearl testosterone-treated mice have significant (P < .001) increases in kidney lysosomal enzyme activity, as compared with normal C57BL/6J mice (Table 3).-Glucuronidase is increased 2-fold in both
mutants as is -galactosidase in pale ear, whereas -galactosidase
is increased 30% in pearl compared to control C57BL/6J. The increases of kidney lysosomal enzymes in double mutant mice were even more pronounced. -Glucuronidase levels were further elevated 1.6-fold (P < .001) over those of either pale ear or pearl, and
-galactosidase levels were 1.4-fold greater (P < .02)
than pale ear. Altogether, -glucuronidase and -galactosidase
concentrations of double mutant mice are increased a striking 3.7- and
2.5-fold, respectively, over that of normal C57BL/6J controls.
Large increases (2.6-fold for There were no significant alterations of liver, brain, or spleen
lysosomal enzymes in either single or double mutants with the
exceptions of small increases in brain The major mechanism for regulation of lysosomal enzyme levels in mouse
kidney is secretion into urine.21 It is obvious from an
analysis of urine levels (Table 4) that
this process is highly abnormal in double mutants and is responsible
for their increased kidney lysosomal enzyme levels. Normal C57BL/6J
mice secreted approximately 15% of total kidney lysosomal enzyme
levels daily. Secretion rates in contrast were about 8%, 40%, and
2.5% (P < .001 for all) of normal rates in ep/ep,
pe/pe, and double mutants, respectively. Secretion rates of double
mutants were 35% (P < .02) of that observed in
ep/ep mutants for both enzymes. Testosterone-mediated induction of kidney lysosomal enzymes is accompanied by large increases
in kidney hypertrophy, and this process is accentuated in mutant mice
with lysosomal enzyme secretion deficits.21 Kidney hypertrophy was in fact most pronounced in pearl and in double mutant
mice (not shown), consistent with a major secretion defect. Mutant
kidney hypertrophy is likely due in large part to an inability to
eliminate by secretion material engorged within lysosomes.
In the mouse, there are at least 15 models for
HPS.4,5 The fact that all these mouse genes for HPS affect
the same subcellular organelles, melanosomes, lysosomes, and platelet
dense granules, indicates that the biosynthesis/function of these 3 organelles are under multiple genetic regulatory controls. Several of
these 15 genes are involved in vesicle trafficking. These include (1) pallid, a novel 25-kd protein that interacts with syntaxin
13,27 (2) gunmetal, the An experimental advantage of the mouse is that it is possible to test for interaction of recessive genes by producing and analyzing offspring that are genetically homozygous for both mutant alleles.16 If the mutations are in a common pathway or within the subunits of a common protein complex, offspring will resemble one or the other parent. If the mutations are in independent pathways, the double mutant is expected to exhibit additive or interactive effects, and a new, usually more severe, phenotype occurs. Clearly, the present data for the pale ear and pearl genes support the latter possibility for nearly all organelle characteristics tested. Both pearl and pale ear are maintained as congenic mutations on a common C57BL/6J inbred background, a fact that greatly simplifies interpretation of new phenotypes because there is no contribution of differing background genes in the double mutant. Also, the molecular nature of each mutation predicts that the function of both proteins is essentially completely abrogated in the double mutant.11,12,15 Thus, there is minimal or no contribution of normal gene products in double mutant mice to confound interpretations. Intercross matings of the pearl and pale ear mutants have produced a
double mutant mouse with new severe phenotypes of platelet dense
granules, lysosomes, and melanosomes re-emphasizing the interrelatedness of these organelles. The platelet effects of HPS
mutations are very selective as they are confined to dense granules and
lysosomes,4 and this pattern was maintained in double HPS
mutants. The production of more severe granule phenotypes is especially
apparent in the case of platelet dense granules where platelet
serotonin levels were depressed to only 1% that of normal platelets.
This is the lowest level documented in any mouse HPS mutant and
predicts a very severe platelet storage pool deficiency and bleeding
phenotype. Thrombin-stimulated platelet lysosomal enzyme secretion was
severely impaired in the double mutant though in this case impairment
was equivalent to that of the single mutant pearl. It remains possible
therefore that the pale ear and pearl genes act within a common pathway
for this process. Mutant effects on platelets are specific to dense
granules and lysosomes. No effects on contents or rates of secretion of the Several lines of evidence support cooperation of the pale ear and pearl genes in the biogenesis of melanosomes. The gross pigmentation phenotype of the double mutant is unlike that of either single mutant. Double mutant mice are conspicuously more hypopigmented in both coat and eye color than either single mutant. The bases of the eye hypopigmentation became apparent upon ultrastructural analyses of the retinal pigment epithelium and choroidal melanocytes of the eye. Most notable was the near absence of pigmentation of the choroid. It is likely that the cause of the pink eye color of the adult double mutant originates in the choroid, because the near absence of RPE melanosomes in pale ear and pearl mice nevertheless produces a dark adult eye color. The observed near absence of RPE melanosomes in the pale ear12,31 and pearl31,32 mutants is consistent with previous observations by others. In agreement with the pigmentation results, the lysosomal phenotype of
the double mutant is considerably more severe than that of either
single mutant. This is apparent in kidney proximal tubule cells where
the constitutive secretion of the soluble lysosomal enzymes
A very interesting lysosomal phenotype of the double mutant, in terms of known clinical features of human HPS, is the increase in lysosomal enzyme concentrations in lung. A significant fraction of patients with HPS1 die in midlife due to lung abnormalities including lung fibrosis.33,34 Similarly reductions in life span and abnormalities of lung structure occur in selected mouse HPS mutants, including pearl35 and pale ear.5 Accumulations of lysosomal ceroid or aging pigment has been postulated as the cause of the lung abnormalities.36 It is possible that increased number or size of lysosomes leads to increased fragility of lysosomes followed by more severe lung abnormalities including death of lung cells. Although further experiments are required to establish the basis of the high lysosomal enzyme concentrations in lung, a reasonable hypothesis is that they may derive, as observed for kidney of the double mutant, from abnormally low secretion rates. This scenario is consistent with the fact that secretion of lysosomal contents is an important process in lungs. The contents, including pulmonary surfactant, of lamellar bodies or lysosomes, are regularly secreted from type II cells in delivery of surfactant to the air-liquid interface to lower surface tension in the lung.37 It is clear that lysosomal enzyme secretion is important in a wide variety of physiologic processes in higher organisms.4,21,38-41 The conclusion that the pale ear and pearl gene products interact indirectly and independently to produce more severe melanosome, lysosome, and platelet dense granule phenotypes is consistent with other recent studies at the cell and molecular levels. For example, the intracellular location of the AP-3 complex is not changed in fibroblasts of patients with HPS17 or in normal fibroblasts overexpressing a His6-HPS1 fusion protein.9 Similarly, the abnormal trafficking of the lysosomal membrane proteins CD63 and LAMP1 commonly observed in HPS2 cells is not observed in HPS1 fibroblasts,9 arguing against a role for the HPS1 protein in the AP-3 dependent pathway for lysosomal membrane protein trafficking. Finally, immunoprecipitation-recapture experiments failed to show an association of the HPS1 protein with AP-3.9 Mice with the new phenotype are viable, breed true, and are by molecular tests doubly homozygous for both the pearl and pale ear recessive genes. Doubly mutant HPS mice provide the practical advantage that they accentuate the mutant phenotype. This may allow, for example, analysis of abnormalities at a considerably earlier age than possible in single mutants, which typically require aging to more than 1 year for appearance of HPS abnormalities such as abnormal lung manifestations.35 The reduced breeding performance of the double mutants suggests that the combined mutant HPS genes have additional general deleterious physiologic effects. This suggestion is consistent with shortened life spans observed with other mouse strains doubly homozygous for other combinations of HPS genes.35 Analyses of double mutants of mouse models for the HPS (pale ear) and Chediak-Higashi (beige) genes similarly concluded that these genes acted on independent pigmentation pathways.42 However, the results of these double mutant experiments clearly differ from those involving double mutants of 2 other mouse HPS mutants, light ear and pale ear.43 In the latter case, the phenotypes of the double mutants were identical to the single mutant parents, suggesting that the pale ear and light ear gene products may directly interact in a common path or common protein complex. Together, these results indicate that the pale ear and pearl genes are functionally interactive at the level of the whole organism in related, yet independent pathways to affect the synthesis/function of specialized mammalian organelles such as melanosomes, lysosomes, and platelet dense granules. The interactive effect of the double mutant on the morphology and physiology of several subcellular organelles is consistent with recent molecular evidence that other mouse genes for HPS, in addition to pale ear and pearl, encode genes that regulate vesicle trafficking. It appears likely that HPS proteins form a physiologic module44 of directly and indirectly interacting proteins necessary for the synthesis of these specialized mammalian organelles.
We thank Aaron Mammoser and Donna Reddington for excellent technical assistance.
Submitted August 24, 2001; accepted October 18, 2001.
Supported by National Institutes of Health grants HL51480, HL31698, and EY12104 (to R.T.S.), by a Medical Research Council program grant awarded to Colin Hopkins, and by the Roswell Park Cancer Institute Cancer Center Support Grant CA 16056.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Richard T. Swank, Molecular and Cellular Biology Dept, Roswell Park Cancer Institute, Carlton and Elm Sts, Buffalo, NY 14263; e-mail: richard.swank{at}roswellpark.org.
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© 2002 by The American Society of Hematology.
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  S. M. Di Pietro, J. M. Falcon-Perez, D. Tenza, S. R.G. Setty, M. S. Marks, G. Raposo, and E. C. Dell'Angelica BLOC-1 Interacts with BLOC-2 and the AP-3 Complex to Facilitate Protein Trafficking on Endosomes Mol. Biol. Cell, September 1, 2006; 17(9): 4027 - 4038. [Abstract] [Full Text] [PDF]
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 S. Fontana, S. Parolini, W. Vermi, S. Booth, F. Gallo, M. Donini, M. Benassi, F. Gentili, D. Ferrari, L. D. Notarangelo, et al. Innate immunity defects in Hermansky-Pudlak type 2 syndrome Blood, June 15, 2006; 107(12): 4857 - 4864. [Abstract] [Full Text] [PDF]
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 S. H. Guttentag, A. Akhtar, J.-Q. Tao, E. Atochina, M. E. Rusiniak, R. T. Swank, and S. R. Bates Defective Surfactant Secretion in a Mouse Model of Hermansky-Pudlak Syndrome Am. J. Respir. Cell Mol. Biol., July 1, 2005; 33(1): 14 - 21. [Abstract] [Full Text] [PDF]
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 C. Haqq, M. Nosrati, D. Sudilovsky, J. Crothers, D. Khodabakhsh, B. L. Pulliam, S. Federman, J. R. Miller III, R. E. Allen, M. I. Singer, et al. From The Cover: The gene expression signatures of melanoma progression PNAS, April 26, 2005; 102(17): 6092 - 6097. [Abstract] [Full Text] [PDF]
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B. Gwynn, J. A. Martina, J. S. Bonifacino, E. V. Sviderskaya, M. L. Lamoreux, D. C. Bennett, K. Moriyama, M. Huizing, A. Helip-Wooley, W. A. Gahl, et al. Reduced pigmentation (rp), a mouse model of Hermansky-Pudlak syndrome, encodes a novel component of the BLOC-1 complex Blood, November 15, 2004; 104(10): 3181 - 3189. [Abstract] [Full Text] [PDF]
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 J. Kaput, K. G. Klein, E. J. Reyes, W. A. Kibbe, C. A. Cooney, B. Jovanovic, W. J. Visek, and G. L. Wolff Identification of genes contributing to the obese yellow Avy phenotype: caloric restriction, genotype, diet x genotype interactions Physiol Genomics, August 11, 2004; 18(3): 316 - 324. [Abstract] [Full Text] [PDF]
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 R. Gautam, S. Chintala, W. Li, Q. Zhang, J. Tan, E. K. Novak, S. M. Di Pietro, E. C. Dell'Angelica, and R. T. Swank The Hermansky-Pudlak Syndrome 3 (Cocoa) Protein Is a Component of the Biogenesis of Lysosome-related Organelles Complex-2 (BLOC-2) J. Biol. Chem., March 26, 2004; 279(13): 12935 - 12942. [Abstract] [Full Text] [PDF]
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 T. A. Lyerla, M. E. Rusiniak, M. Borchers, G. Jahreis, J. Tan, P. Ohtake, E. K. Novak, and R. T. Swank Aberrant lung structure, composition, and function in a murine model of Hermansky-Pudlak syndrome Am J Physiol Lung Cell Mol Physiol, September 1, 2003; 285(3): L643 - L653. [Abstract] [Full Text] [PDF]
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R. Nazarian, J. M. Falcon-Perez, and E. C. Dell'Angelica Biogenesis of lysosome-related organelles complex 3 (BLOC-3): A complex containing the Hermansky-Pudlak syndrome (HPS) proteins HPS1 and HPS4 PNAS, July 22, 2003; 100(15): 8770 - 8775. [Abstract] [Full Text] [PDF]
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M. L. Klebig, M. D. Wall, M. D. Potter, E. L. Rowe, D. A. Carpenter, and E. M. Rinchik Mutations in the clathrin-assembly gene Picalm are responsible for the hematopoietic and iron metabolism abnormalities in fit1 mice PNAS, July 8, 2003; 100(14): 8360 - 8365. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
Th) and thrombin (+Th)-treated platelets of mutant and normal mice.
After treatment of platelets for 3 minutes with buffer or 1.6 U/mL
thrombin, platelets were centrifuged and equivalent proportions of
pellets (P) and supernatants (S) were electrophoresed on denaturing
gels that were immunoblotted with antifibrinogen antibody. (B)
Steady-state levels of PF4 in normal and mutant platelets. Platelet
protein (30 µg) was electrophoresed on denaturing gels and
immunoblotted with antibody to PF4.
subunit of the AP-3
complex,