The gene mutated in cocoa mice, carrying a defect of organelle biogenesis, is a homologue of the human Hermansky-Pudlak syndrome-3 gene.

Hermansky-Pudlak syndrome (HPS) is a group of human disorders of organelle biogenesis characterized by defective synthesis of melanosomes, lysosomes, and platelet dense granules. In the mouse, at least 15 loci are associated with mutant phenotypes similar to human HPS. We have identified the gene mutated in cocoa (coa) mice, which is associated with an HPS-like mutant phenotype and thus represents a strong candidate for human HPS. Analysis of coa-mutant mice and cultured coa-mutant mouse melanocytes indicates that the normal coa gene product is involved in early stages of melanosome biogenesis and maturation.

Genomic structure of the mouse Ap3b1 gene in normal and pearl mice.

The mouse hypopigmentation mutant pearl is an established model for Hermansky-Pudlak syndrome (HPS), a genetically heterogenous disease with misregulation of the biogenesis/function of melanosomes, lysosomes, and platelet dense granules. The pearl (Ap3b1) gene encodes the beta3A subunit of the AP-3 adaptor complex, which regulates vesicular trafficking. The genomic structure of the normal Ap3b1 gene includes 25 introns and a putative promoter sequence. The original pearl (pe) mutation, which has an unusually high reversion rate on certain strain backgrounds, has been postulated to be caused by insertion of a transposable element. Indeed, the mutation contains a 215-bp partial mouse transposon at the junction point of a large tandem genomic duplication of 6 exons and associated introns. At the cDNA level, three pearl mutations (pearl, pearl-8J, and pearl-9J) are caused by deletions or duplications of a complete exon(s).

Abnormal vesicular trafficking in mouse models of Hermansky-Pudlak syndrome.

Hermansky-Pudlak Syndrome (HPS) is a group of related multigenic recessively inherited disorders which causes abnormalities in the biosynthesis and/or function of three related organelles; melanosomes, platelet-dense granules and lysosomes. These lead, in turn, to hypopigmentation, prolonged bleeding and ceroid deposition. Positional cloning strategies have identified five mouse HPS genes. Two orthologous human diseases (HPS1 and HPS2) have likewise been identified. At least four of the five mouse genes encode proteins involved in the regulation of intracellular vesicle trafficking. The pearl (HPS2) and mocha genes encode the beta3A and delta subunits, respectively, of the AP-3 adaptor complex, which captures organelle membrane proteins at the trans-Golgi apparatus. The protein products of the pallid and gunmetal genes are also important components of the vesicular trafficking machinery. The former interacts with a t-SNARE, syntaxin13, and the latter is the alpha subunit of Rab geranylgeranyltransferase, which renders Rab proteins sufficiently lipophilic to function at their target membranes. The pale ear (HPS1) gene encodes a ubiquitously expressed protein of unknown function. Recent physiological studies have shown that mouse HPS mutants, like their human HPS counterparts, have variably reduced lifespans and may have lung abnormalities.

A mutation in Rab27a causes the vesicle transport defects observed in ashen mice.

The dilute (d), leaden (ln), and ashen (ash) mutations provide a unique model system for studying vesicle transport in mammals. All three mutations produce a lightened coat color because of defects in pigment granule transport. In addition, all three mutations are suppressed by the semidominant dilute-suppressor (dsu), providing genetic evidence that these mutations function in the same or overlapping transport pathways. Previous studies showed that d encodes a major vesicle transport motor, myosin-VA, which is mutated in Griscelli syndrome patients. Here, using positional cloning and bacterial artificial chromosome rescue, we show that ash encodes Rab27a. Rab GTPases represent the largest branch of the p21 Ras superfamily and are recognized as key players in vesicular transport and organelle dynamics in eukaryotic cells. We also show that ash mice have platelet defects resulting in increased bleeding times and a reduction in the number of platelet dense granules. These defects have not been reported for d and ln mice. Collectively, our studies identify Rab27a as a critical gene for organelle-specific protein trafficking in melanocytes and platelets and suggest that Rab27a functions in both MyoVa dependent and independent pathways.

Rab geranylgeranyl transferase alpha mutation in the gunmetal mouse reduces Rab prenylation and platelet synthesis.

Few molecular events important to platelet biogenesis have been identified. Mice homozygous for the spontaneous, recessive mutation gunmetal (gm) have prolonged bleeding, thrombocytopenia, and reduced platelet alpha- and delta-granule contents. Here we show by positional cloning that gm results from a G-->A substitution mutation in a splice acceptor site within the alpha-subunit of Rab geranylgeranyl transferase (Rabggta), an enzyme that attaches geranylgeranyl groups to Rab proteins. Most Rabggta mRNAs from gm tissues skipped exon 1 and lacked a start codon. Rabggta protein and Rab geranylgeranyl transferase (GGTase) activity were reduced 4-fold in gm platelets. Geranylgeranylation and membrane association of Rab27, a Rab GGTase substrate, were significantly decreased in gm platelets. These findings indicate that geranylgeranylation of Rab GTPases is critical for hemostasis. Rab GGTase inhibition may represent a new treatment for thrombocytosis and clotting disorders.

Porphyromonas gingivalis platelet aggregation activity: outer membrane vesicles are potent activators of murine platelets.

Recent evidence has established an association between chronic periodontitis and cardiovascular disease. Periodontitis is a chronic inflammatory disease caused by a small group of gram-negative bacteria, of which Porphyromonas gingivalis is considered an important causative agent. It has been proposed that dental plaque bacteria and their products can disseminate into the bloodstream from the site of infection and promote thromboembolic events associated with atherosclerosis and myocardial infarction. In this regard, Streptococcus sanguis and P. gingivalis have been shown to induce platelet aggregation in vitro. Here we report that P. gingivalis was able to induce platelet aggregation, and that oral strains of Actinobaillus actinomycetemcomitans, Bacteroides forsythus, Campylobacter rectus, Fusobacterium nucleatum, Prevotella intermedia and Trepenoma denticola failed to aggregate platelets when tested for platelet aggregation activity under similar conditions. Additionally, we show that vesicles (outer membrane evaginations that are shed into the environment by the bacteria) of P. gingivalis are potent inducers of mouse platelet aggregation in vitro. In summary, our data show that i) initial adherence of the bacterium to platelet may be facilitated by P. gingivalis fimbriae and ii) P. gingivalis vesicles possess platelet aggregation-inducing activity.

5'-UTR structural organization, transcript expression, and mutational analysis of the human Rab geranylgeranyl transferase alpha-subunit (RABGGTA) gene.

Hermansky-Pudlak syndrome (HPS) is a recessively inherited disease with dysfunction of several related subcellular organelles including platelet-dense granules, melanosomes, and lysosomes. Our recent identification of the mutation in murine Rab geranylgeranyl transferase alpha-subunit gene (Rabggta) in one mouse model of HPS, the gunmetal mouse, suggested that human patients with similar phenotypes might have mutations in the human orthologous RABGGTA gene. This prompted reanalysis of the 5'-untranslated structure of the human RABGGTA gene in normal individuals and in patients with deficiencies of platelet-dense granules (alphadelta-SPD), alpha granules (alpha-SPD or gray platelet syndrome, GPS) or alpha plus dense granules (alphadelta-SPD). We report the complete sequence of intron alpha of RABGGTA and demonstrate that exon alpha is immediately upstream of intron alpha. The exon/intron structural organization of the 5'-untranslated region (UTR) of human RABGGTA was found to be similar to that of the mouse Rabggta gene. However, exons alpha and introns alpha are not homologous between mouse and human. Features of the 5'-UTR of RABGGTA suggest it is a housekeeping gene. While obvious disease-causing mutations of human RABGGTA were not found in our existing SPD patients by sequencing its entire coding region, several polymorphisms of RABGGTA including a putative cryptic splicing mutation in intron 4 were identified. Knowledge of the 5'-UTR structure of RABGGTA and its common polymorphisms will be useful for mutation screening or linkage analysis in patients with albinism, thrombocytopenia, or platelet SPD.

Analyses of proteins involved in vesicular trafficking in platelets of mouse models of Hermansky Pudlak syndrome.

Hermansky Pudlak syndrome (HPS) is an autosomal recessive inherited disorder characterized by defects in synthesis and/or secretion of three related subcellular organelles: melanosomes, platelet-dense granules, and lysosomes. In the mouse, mutant forms of any of 14 separate genes result in an HPS-like phenotype. The mouse pearl and mocha genes encode subunits of the AP3 adaptor protein complex, confirming that HPS mutations involve proteins regulating intracellular vesicular trafficking. Therefore, expression of several additional proteins involved in vesicular transport was examined by immunoblotting of platelet extracts from HPS mutant and control mice. Platelet levels of SCAMPS (secretory carrier membrane proteins), Rab11, Rab31, NSF (N-ethylmaleimide-sensitive fusion protein), syntaxin 2, syntaxin 4, munc18c, and p115/TAP (p115/transcytosis-associated protein) were not significantly altered in several different HPS mutants. However, gunmetal (gm/gm) platelets contained decreased amounts of SNAP-23. The Snap23 gene was mapped to mouse chromosome 5, demonstrating it cannot encode the gm gene, which maps to chromosome 14. It is likely therefore that the gm gene functions upstream of SNAP-23 in vesicular trafficking.

Increased incidence and analysis of emperipolesis in megakaryocytes of the mouse mutant gunmetal.

Mutant gunmetal (gm/gm) mice exhibit prolonged bleeding, platelet granule defects, abnormal megakaryocyte demarcation membranes, and thrombocytopenia. The number of megakaryocytes in gm/gm mice is increased substantially. Also, the percentage of gm/gm megakaryocytes exhibiting emperipolesis is increased. However, the number of emperipolesed cells per megakaryocyte is not. EC are of several hematopoietic lineages, with a slight skew to granulocytes, and include mature, primitive, and degenerating cells. No significant differences in the types of emperipolesed cells were observed between mutant mice and their normal gm/+ or +/+ counterparts. The increased incidence of emperipolesis in gm/gm megakaryocytes is controlled by the megakaryocyte genotype, not systemic factors. A significant practical finding of these studies was the demonstration that increased emperipolesis results in a significant "right shift" in megakaryocyte ploidy determined by flow cytometry.

Hygienic mycology in Hungary.

In Hungary the medical mycological research concerning the systemic (so-called deep) mycoses started in the National Institute of Hygiene. The Mycology Department has been founded in 1955 in this Institute with nation-wide authority as a routine diagnostic laboratory for clinical specimens from systemic mycotic infections. Three years later its activity was broadened--on ground of the experiences--to cover the total field of hygiene, thus we had to establish the "mycologia hygienica" (hygienic mycology), however, this has not been accepted as an independent discipline. Here a short, selected overview is given of our experiences. In the following the trends of the activity is demonstrated along the subdivisions of hygiene (epidemiology, environmental-hygiene, professional and occupational-, nutritional and food hygiene). In respect of hygiene, however, the fungi play a Janus-faced (more exactly Dr. Jekyll-Mr. Hyde) role, as they can behave not only harmful but also beneficial.

Abnormal expression and subcellular distribution of subunit proteins of the AP-3 adaptor complex lead to platelet storage pool deficiency in the pearl mouse.

The pearl mouse is a model for Hermansky Pudlak Syndrome (HPS), whose symptoms include hypopigmentation, lysosomal abnormalities, and prolonged bleeding due to platelet storage pool deficiency (SPD). The gene for pearl has recently been identified as the beta3A subunit of the AP-3 adaptor complex. The objective of these experiments was to determine if the expression and subcellular distribution of the AP-3 complex were altered in pearl platelets and other tissues. The beta3A subunit was undetectable in all pearl cells and tissues. Also, expression of other subunit proteins of the AP-3 complex was decreased. The subcellular distribution of the remaining AP-3 subunits in platelets, macrophages, and a melanocyte-derived cell line of pearl mice was changed from the normal punctate, probably endosomal, pattern to a diffuse cytoplasmic pattern. Ultrastructural abnormalities in mutant lysosomes were likewise apparent in mutant kidney and a cultured mutant cell line. Genetically distinct mouse HPS models had normal expression of AP-3 subunits. These and related experiments strongly suggest that the AP-3 complex regulates the biogenesis/function of organelles of platelets and other cells and that abrogation of expression of the AP-3 complex leads to platelet SPD.

Survival and lung pathology of mouse models of Hermansky-Pudlak syndrome and Chediak-Higashi syndrome.

Hermansky-Pudlak Syndrome (HPS), a recessively inherited disease in humans, affects the biosynthesis/processing of the related intracellular organelles: lysosomes, melanosomes, and platelet dense granules. The disease is multigenic in both humans and mice where 14 separate genes have been demonstrated to be causative. Patients often die prematurely with severe lung abnormalities. Patients with the related Chediak-Higashi Syndrome (CHS) likewise have significantly reduced life spans. Long-term survival and lung histomorphology were analyzed in a pilot experiment involving several genetically defined singly and doubly mutant mouse HPS mutants and the beige CHS mutant to determine whether these parameters are altered in the mouse models. The mutants differed widely in both longevity and lung architecture. Mice doubly homozygous for the pale ear and ruby eye or for the muted and pearl genes had the shortest life spans with none surviving the two-year experimental duration. Life spans were similarly severely reduced in the beige and gunmetal mutants. Intermediate life spans were apparent in the pearl, pallid, and cocoa mutants whereas minimal effects were noted in ruby eye, muted, light ear, and cocoa mutants. Enlarged air spaces were noted in histologic sections of lungs of several of the mutants. For the most part, the severity of lung abnormalities was inversely proportional to the long-term survival of these various mutants, suggesting that lung pathology may contribute to mortality, as has been suggested for human HPS patients.

The beta3A subunit gene (Ap3b1) of the AP-3 adaptor complex is altered in the mouse hypopigmentation mutant pearl, a model for Hermansky-Pudlak syndrome and night blindness.

Lysosomes, melanosomes and platelet-dense granules are abnormal in the mouse hypopigmentation mutant pearl. The beta3A subunit of the AP-3 adaptor complex, which likely regulates protein trafficking in the trans - Golgi network/endosomal compartments, was identified as a candidate for the pearl gene by a positional/candidate cloning approach. Mutations, including a large internal tandem duplication and a deletion, were identified in two respective pearl alleles and are predicted to abrogate function of the beta3A protein. Significantly lowered expression of altered beta3A transcripts occurred in kidney of both mutant alleles. The several distinct pearl phenotypes suggest novel functions for the AP-3 complex in mammals. These experiments also suggest mutations in AP-3 subunits as a basis for unique forms of human Hermansky-Pudlak syndrome and congenital night blindness, for which the pearl mouse is an appropriate animal model.

cDNA sequence and mapping of the mouse Copb gene encoding the beta subunit of the COPI coatomer complex.

COPI-coated vesicles are involved in retrograde-directed selective transport of proteins from the Golgi complex to the endoplasmic reticulum (ER) as well as mediate anterograde transport of cargo proteins within the Golgi or in endosomal trafficking. The COPI protein complex contains an ADP-ribosylation factor (ARF1) and seven coatamer subunits (alpha, beta, beta', gamma, delta, epsilon, zeta-COP). The localization and function of human beta subunit of coatamer (COPB) suggests it is likely a candidate gene of ruby-eye-2 (ru2), which is a mouse model of human Hermansky-Pudlak syndrome characterized by the dysfunction of several subcellular organelles. In this study, we determined the entire coding sequence of mouse (Copb) cDNA by combining an overlapping mouse EST contig with EST walking. beta-COP was found highly conserved in mouse, rat, and human, and it is ubiquitously expressed in mouse. The Copb gene was mapped to mouse Chr 7 at a position of 53.3 cM by radiation hybrid mapping. Our RH mapping data, sequencing of RT-PCR products, and Western blotting exclude the Copb gene as a candidate for ru2.

Mouse models of Hermansky Pudlak syndrome: a review.

Hermansky Pudlak Syndrome (HPS) is a recessively inherited disease affecting the contents and/or the secretion of several related subcellular organelles including melanosomes, lysosomes, and platelet dense granules. It presents with disorders of pigmentation, prolonged bleeding, and ceroid deposition, often accompanied by severe fibrotic lung disease and colitis. In the mouse, the disorder is clearly multigenic, caused by at least 14 distinct mutations. Studies on the mouse mutants have defined the granule abnormalities of HPS and have shown that the disease is associated with a surprising variety of phenotypes affecting many tissues. This is an exciting time in HPS research because of the recent molecular identification of the gene causing a major form of human HPS and the expected identifications of several mouse HPS genes. Identifications of mouse HPS genes are expected to increase our understanding of intracellular vesicle trafficking, lead to discovery of new human HPS genes, and suggest diagnostic and therapeutic approaches toward the more severe clinical consequences of the disease.

The murine misty mutation: phenotypic effects on melanocytes, platelets and brown fat.

Although the recessive murine mutation misty (m) is well known, its phenotype has never been reported beyond brief descriptions of a dilution of coat color and white spotting of the belly and extremities, suggesting a developmental mutation. A report in abstract has also suggested effects on white fat and body weight. Here, we report effects of the homozygous misty mutation on an unusual combination of three cell types: melanocytes, platelets, and brown fat. Brown fat appeared to be completely absent from all expected locations in neonatal m/m mice. A prolonged bleeding time was observed; platelet count and platelet serotonin and ATP levels were normal, but the level of ADP in m/m platelets was low. Primary cultures and immortal lines of melanocytes from m/m mice showed several abnormalities. There was a marked deficiency in net proliferation, suggesting that the color dilution and spotting in vivo may result from reduced numbers of melanocytes and their precursors. m/m melanocytes were also hyperdendritic in morphology, overproduced melanin, and had deficient responses to the cAMP agonists cholera toxin and melanocyte-stimulating hormone, which normally promote melanin production. The misty gene product may be involved in adenine nucleotide metabolism or signaling.

Analysis of the Kit and Pdgfra genes in the patch-extended (Phe) mutation.

The patch (Ph) locus allele, patch-extended (Phe), has significantly less pigmentation than the original mutation and homozygotes have been known to survive to term. Analysing intersubspecific F1 hybrids, we were able to demonstrate that Phe is a deletional mutation encompassing the platelet-derived growth factor receptor alpha subunit (Pdgfra). The deletion does not appear to extend into the coding sequence of the Kit gene (a related tyrosine kinase receptor). However, we were able to demonstrate that, while the Kit gene is transcribed, it does not encode a functionally active receptor.

The mouse pale ear (ep) mutation is the homologue of human Hermansky-Pudlak syndrome.

The recessive mutation at the pale ear (ep) locus on mouse chromosome 19 was found to be the homologue of human Hermansky-Pudlak syndrome (HPS). A positional cloning strategy using yeast artificial chromosomes spanning the HPS locus was used to identify the HPS gene and its murine counterpart. These genes and their predicted proteins are highly conserved at the nucleotide and amino acid levels. Sequence analysis of the mutant ep gene revealed the insertion of an intracisternal A particle element in a protein-coding 3' exon. Here we demonstrate that mice with the ep mutation exhibit abnormalities similar to human HPS patients in melanosomes and platelet-dense granules. These results establish an animal model of HPS and will facilitate biochemical and molecular analyses of the functions of this protein in the membranes of specialized intracellular organelles.

Biosynthesis of renin in mouse kidney tumor As4.1 cells.

As4.1, a renin-expressing cell line isolated from a mouse renal tumor, was characterized for synthesis, processing, storage and secretion of renin polypeptides. Metabolic labeling, immunoprecipitation and SDS/PAGE analysis revealed that renin was secreted into the culture supernatant predominantly in the form of prorenin which migrated as products of 42-47 kDa. The predominant intracellular renin was processed into two chains, of 33-34 and 5 kDa. N-glycanase treatment removed N-linked oligosaccharides and yielded products of 41 kDa for prorenin and 31-32 kDa for the heavier chain of two-chain renin. The N-terminus of the constitutively secreted prorenin was determined by automated Edman degradation to be Leu22 while the N-terminus of the heavy chain was Ser72. Renin polypeptides constituted 3.1 +/- 1.4% (mean percentage of total precipitable radioactivity +/- SD) of de-novo-synthesized protein secreted into the medium and 0.2 +/- 0.17% retained intracellularly. Extrapolation of renin activity assays suggest that a single cell stores approximately 680 fg of active renin. A slow incremental release into the medium of processed renin heavy chain was detected by immunoprecipitation and SDS/PAGE. Renin activity assays confirmed the release of approximately 4 fg prorenin and 0.32 fg active renin cell(-1) h(-1). Indirect immunofluorescence demonstrated intracellular renin to be distributed in a punctate pattern. Renin was found to be colocalized with the lysosomal marker, beta-glucuronidase, by double-fluorescent labeling. These cells have enabled characterization of glycosylated mouse renin-1 and may prove a valuable tool for studying intracellular trafficing of renin and associated processing enzymes.

A novel modifier gene for plasma von Willebrand factor level maps to distal mouse chromosome 11.

Type 1 von Willebrand disease (VWD), characterized by reduced levels of plasma von Willebrand factor (VWF), is the most common inherited bleeding disorder in humans. Penetrance of VWD is incomplete, and expression of the bleeding phenotype is highly variable. In addition, plasma VWF levels vary widely among normal individuals. To identify genes that influence VWF level, we analyzed a genetic cross between RIIIS/J and CASA/Rk, two strains of mice that exhibit a 20-fold difference in plasma VWF level. DNA samples from F2 progeny demonstrating either extremely high or extremely low plasma VWF levels were pooled and genotyped for 41 markers spanning the autosomal genome. A novel locus accounting for 63% of the total variance in VWF level was mapped to distal mouse chromosome 11, which is distinct from the murine Vwf locus on chromosome 6. We designated this locus Mvwf for "modifier of VWF." Additional genotyping of as many as 2407 meioses established a high resolution genetic map with gene order Cola1-Itg3a-Ngfr-Mvwf/Gip-Hoxb9-Hoxb1++ +-Cbx'rs2-Cox5a-Gfap. The Mvwf candidate interval between Ngfr and Hoxb9 is approximately 0.5 centimorgan (cM). These results demonstrate that a single dominant gene accounts for the low VWF phenotype of RIIIS/J mice in crosses with several other strains. The pattern of inheritance suggests a gain-of-function mutation in a unique component of VWF biosynthesis or processing. Characterization of the human homologue for Mvwf may have relevance for a subset of type 1 VWD cases and may define an important genetic factor modifying penetrance and expression of mutations at the VWF locus.