Genetic and molecular control of folate-homocysteine metabolism in mutant mice.

Hyperhomocysteinemia adversely affects fundamental aspects of fetal development, adulthood, and aging, but the role of elevated homocysteine levels in these birth defects and adult diseases remains unclear. Mouse models are valuable for investigating the causes and consequences of hyperhomocysteinemia. We used a phenotype-based approach to identify mouse mutants for studying the relation between single gene mutations, homocysteine levels as a measure of the status of homocysteine metabolism, and gene expression profiles as a way to assess the impact of protein deficiency in mutant mice on steady-state transcription levels of genes in the folate-homocysteine pathways. These mutants were selected based on their propensity to produce phenotypes that are reminiscent of those associated with anomalies in folate-homocysteine metabolism in humans. We report identification of new, single-gene mouse models of homocysteinemia and characterization of their molecular and physiological impact on folate-homocysteine metabolism. Mutations in several genes involved in the hedgehog and WNT signal transduction pathways, as well as a gene involved in lipid metabolism, resulted in elevated homocysteine levels and altered expression profiles of folate-homocysteine metabolism genes. These results begin to unravel the complex relations between elevation of a single amino acid in the blood and the diverse birth defects and adult diseases associated with hyperhomocysteinemia.

Reduced incidence of thrombosis in mice with hereditary spherocytosis following neonatal treatment with normal hematopoietic cells.

Thrombosis is a life-threatening complication of hemolytic anemia in humans. Cardiac thrombi are present in all adult alpha-spectrin-deficient (sph/sph) mice with severe hereditary spherocytosis, providing a model for events preceding thrombosis. The current study evaluated (1) the timing of thrombosis initiation and (2) the effect of postnatal transplantation of normal cells on life span and thrombotic incidence in adult mice. Thrombi are detected histologically following necropsy in untreated sph/sph mice of various ages and are not observed until 6 weeks of age. Thrombotic incidence increases from 50% at 6 to 7 weeks of age to 100% at 9 weeks of age. As a potential therapy, nonablated sph/sph neonates were transfused with either genetically marked normal peripheral blood (PB), bone marrow (BM), or both and assessed for donor cells and thrombosis. A single transfusion of PB, with or without BM, significantly increases the percentage of sph/sph mice that survive to weaning (4 weeks of age). Replacement in all sph/sph recipients is limited to red blood cells (RBCs). RBCs derived from donor PB are lost within 5 weeks. PB plus BM prolongs high-level donor PB cell production better than BM alone. Thrombotic incidence is significantly reduced in all sph/sph mice treated with PB, BM, or both. Hence, the presence of normal blood cells in the peripheral circulation of neonatal and adult sph/sph mice rescues the former and abrogates the development of thrombosis in the latter. (Blood. 2001;97:3972-3975)

Special considerations in the evaluation of the hematology and hemostasis of mutant mice.

The study of mutant mice with altered or deficient hematopoietic or hemostatic gene products provides a challenge to the researcher, particularly when genetic alterations lead to lethal phenotypes. The following review provides a framework for understanding murine hematopoiesis, based on work with mutant mice, and details experimental approaches used to evaluate these animals. Mice with deficiencies in hemostatic and fibrinolytic system proteins are discussed, and the investigation of their phenotypes is reviewed.

Granulocyte/macrophage colony-stimulating factor and interleukin-3 correct osteopetrosis in mice with osteopetrosis mutation.

Although young mice homozygous for the osteopetrosis (op) mutation usually developed prominent osteopetrosis, its severity was markedly reduced in aged op/op mice. This age-associated reversal of osteopetrosis was accompanied by the expansion of bone marrow cavities and increased numbers of tartrate-resistant acid phosphatase (TRAP)-positive cells and of macrophages in the bone marrow. The TRAP-positive cells were mononuclear and developed ruffled borders and numerous vesicles, vacuoles, and granules. Enzyme-linked immunosorbent assay demonstrated a significant elevation of serum granulocyte/ macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-3 levels in the aged op/op mice. To examine whether GM-CSF and/or IL-3 could correct osteopetrosis in young op/op mice, 5 ng of recombinant murine (rm)GM-CSF and/or 100 ng of rmIL-3 were injected daily into young op/op mice. In these treated young op/op mice, the bone marrow cavities were expanded significantly at 2 weeks after administration, associated with significantly increased numbers of TRAP-positive cells and bone marrow macrophages. TRAP-positive cells increased in number with days after injection. These results suggest that GM-CSF and IL-3 induce the development of osteoclasts to correct osteopetrosis in the op/op mice with aging.

A rhino mutant originating in KK mice.

In 1991, several hairless offspring were found in our black haired KK (KK-C) mouse colony. This mutant, provisionally naming KK-rhino mouse was clinicopathologically and histopathologically investigated in a chronological manner. KK-rhino has a hairless trait and wrinkled and pimpled skin, which resemble the characteristics of the so-called rhino mouse. It was presumed from a preliminary mating examination that the trait of the KK-rhino mutant might be manifested by the [hrrh] gene. Other properties of this mutant might be inherited from the maternal KK-C line. KK-rhino mouse is a unique mouse strain which has two quite different traits, "rhino" and "diabetes".

Concentration of free growth hormone-binding protein in the serum of mice is not regulated by growth hormone.

Ames dwarf mice that do not express growth hormone (GH) or prolactin (PRL) genes were used to study the effects of GH deficiency on the presence and the characteristics of GH-binding protein (GHBP) in serum. Chromatographic techniques were used to allow characterization of biological rather than immunological activity of GHBP. Two GH-binding fractions were found in dwarf mice serum, one with low affinity and high capacity (GHBPI) and one with high affinity, low capacity and lower molecular mass (GHBPII). Serum concentration of the high-affinity GHBP was 0.73 +/- 0.03 nM with a Kd of 6.3 +/- 1.7 nM. Since Ames dwarf mice have no GH in the circulation, all the GHBP is free. Interestingly, the concentration of GHBP in dwarf mice was similar to the levels of free GHBP measured in normal mice from the same line. Moreover, this value (0.7 nM) closely resembles the concentration of free GHBP in the serum of transgenic mice overexpressing GH, in which peripheral GH levels are grossly elevated. These observations can be interpreted as evidence that the levels of free GHBP in mouse serum are independent of GH concentration, and that GH influences only the levels of bound GHBP in peripheral circulation.

Primary structure of murine red blood cell-type pyruvate kinase (PK) and molecular characterization of PK deficiency identified in the CBA strain.

To clarify the molecular abnormality of pyruvate kinase (PK) deficiency identified in the mutant mice of CBA-Pk-1slc/Pk-1slc, we cloned murine red blood cell-type PK (R-PK) cDNA of those animals. The cDNA sequence spans 1827 bp, including an open reading frame that can encode 574 amino acids. Homology in the coding sequences between murine and human R-PK was 86.1% at nucleotide and 91.5% at amino acid levels. A homozygous missense mutation at nucleotide 1013 GGT-->GAT was identified in the cDNA sequence of the mutant, causing a single amino acid substitution at no. 338Gly-->Asp of the murine R-PK. Six amino acid residues, 335Val-336Ala-337Arg-338Gly-339Asp-340L eu, were encoded in exon 8 of both human and rat L (liver-type)/R-PK genes and were evolutionarily conserved in PK from bacteria through humans. 337Arg was reported to be important for substrate binding, suggesting that the amino acid change would impair substrate affinity of the PK subunit. A homozygous missense mutation at the catalytic domain has been identified in a human PK variant, PK Hong Kong (941ATT-->ACT, 314 Ile-->Thr). Although both 1013A and 941C gave rise to an amino acid change adjacent to the active site and may interfere with substrate binding to the subunit, the degree of anemia was much more severe in the human case. The erythroid-progenitor cell number increased in the spleen of Pk-1slc/Pk-1slc mice to a level approximately 66 times higher than that in normal CBA mice, suggesting that compensatory extramedullary erythropoiesis in the spleen of the mutant mice, but not in the human variant, might account for the observed difference in the phenotype.

The cell proliferation-associated protein Ki-67 is a target of autoantibodies in the serum of MRL mice.

BACKGROUND: Ab in the serum of patients with autoimmune diseases have been used to identify, characterize, and purify many autoantigens. EXPERIMENTAL DESIGN: Serum from a patient (Ge) with Sjögren's syndrome was used to identify cDNA clones encoding novel autoantigens. This patient's serum was chosen for study because it contained antinuclear Ab that were different from those frequently detected in patients with autoimmune diseases. RESULTS: Ge serum identified a cDNA clone encoding part of protein Ki-67, a cell proliferation-associated protein. The Ki-67 protein (pKi-67) was not previously known to be a target of autoantibodies. To investigate the association between Ab directed against pKi-67 and autoimmune diseases, sera from autoimmune mice were tested for reactivity with a recombinant fragment of pKi-67. Ab were detected in serum from MRL/MpJ(-)+/+ and MRL/MpJ-lpr/lpr mice but not in serum from other autoimmune mice or control animals. CONCLUSIONS: Protein Ki-67 joins the proliferating cell nuclear Ag (PCNA) as an example of a cell proliferation-associated protein that is a target of autoantibodies. The presence of anti-pKi-67 Ab in MRL mice, but not other autoimmune mice, suggests that anti-pKi-67 Ab may be specific markers for the systemic lupus erythematosus-like illness that develops in these animals. Further characterization of the immune response directed against this Ag may provide clues to the etiology and pathogenesis of autoimmune disease in these animals.

Characterization of plasma lipids in genetically obese mice: the mutants obese, diabetes, fat, tubby, and lethal yellow.

Plasma lipid levels were measured in control strains C57BL/6J (B6) and C57BL/KsJ (BKs) and in the mutants obese (ob), diabetes (db), fat (fat), tubby (tub), and lethal yellow (Ay), which are considered models of non-insulin-dependent diabetes mellitus (NIDDM), to determine if perturbations in plasma lipids were similar to those observed in the obese or diabetic human population. Compared with control mice, obese, diabetes, tubby, and lethal yellow mice had triglyceride levels that were elevated 1.5-fold to twofold, but fat mice had triglyceride levels similar to those of controls. Elevated plasma cholesterol levels, which were also observed in most mutant mice, were mainly due to an increase in high-density lipoprotein cholesterol (HDL-C). The degree of hypercholesterolemia appeared to be related to the age of onset and severity of the obesity and diabetes phenotype, with the greatest elevations occurring in obese and diabetes, milder elevations in fat mice of both sexes, male tubby, and male yellow mice, and no apparent changes in female tubby or lethal yellow mice. Plasma HDL-C and glucose levels and body weight in B6-db/db mice and their normal littermates were measured at intervals between 2 and 12 weeks of age to determine when the changes in cholesterol occurred in relationship to hyperglycemia and obesity. An elevation in HDL-C in B6-db/db mice was apparent by 3 weeks of age, a time concurrent with the elevation in blood glucose but before any weight differences.(ABSTRACT TRUNCATED AT 250 WORDS)

Atherosclerosis in genetically obese mice: the mutants obese, diabetes, fat, tubby, and lethal yellow.

Mice with five different mutations conferring an obese or diabetic phenotype were evaluated for fatty streak lesions after consuming an atherogenic diet containing 15% fat and 1.25% cholesterol (wt/wt) for 14 weeks. The five mutations, fat, obese, tubby, diabetes, and lethal yellow, are maintained as congenic strains with C57BL/6J (B6) or C57BL/KsJ (BKs) as genetic backgrounds. None of the mutants exhibited accelerated fatty streak lesion formation; the mutant fat had aortic lesions comparable in size to those of its control strain, and the mutants obese, diabetes, tubby, and lethal yellow had significantly reduced lesion area in comparison to controls. Although B6 and BKs are closely related strains, we observed that the BKs strain was more prone to early-stage atherogenesis. Fatty streak lesion area was twice as large in BKs mice than those found in B6 mice; likewise, in comparison, the mutants obese and diabetes had larger lesions if they were carried as congenic strains in the BKs rather than the B6 genetic background. Plasma triglycerides, total cholesterol, high-density lipoprotein cholesterol (HDL-C), and combined low-density and very-low-density lipoprotein cholesterol (LDL-C and VLDL) levels were also measured in the mice. Lipid profiles differed among the mutant mice, but in general, elevations in plasma total cholesterol, triglycerides, and HDL-C were observed. Whereas the hypertriglyceridemia and hypercholesterolemia are consistent with an atherogenic lipid profile, HDL-C levels, which are normally decreased in individuals with non-insulin-dependent diabetes mellitus, were increased in the mouse mutants.(ABSTRACT TRUNCATED AT 250 WORDS)

Compensatory mechanisms in platelet production: lack of a paracrine response in W/Wv mice treated with 5-fluorouracil.

W/Wv mice maintain normal platelet levels despite having a reduced functional stem cell pool, indicating that platelet production in these mice is compensated by altered megakaryocytopoiesis. In this study the effect of 5-fluorouracil (5-FU) treatment on platelet production in W/Wv mice and their congenic normal littermates was assessed. Recovery of circulating platelet levels occurred 11 days after 5-FU administration in W/Wv mice and subsequently did not increase above control values. In contrast, normal littermates showed an increased platelet count by day 8 and significant thrombocytosis between days 11 and 14. Investigation of bone marrow megakaryocytopoiesis in W/Wv mice showed there was no recovery in the number of megakaryocyte progenitors (CFU-Meg) per femur between days 3 and 5, but control values were reached by day 10. In addition, by day 8 the number of mature megakaryocytes per unit volume of bone marrow in these mice had not returned to control values, although the megakaryocytes were of an increased size. In comparison, the number of CFU-Meg per femur in normal mice treated with 5-FU began to recover after day 3, returned to control values by day 8 and increased to supranormal levels by day 14. Bone marrow megakaryocyte concentration was increased 2-fold over the control by day 8 and an increase in mean megakaryocyte size was also observed. The data suggest that platelet production in mice is dependent on the rate of establishment of both the progenitor cell and megakaryocyte pools. The inability of W/Wv mice to enhance and accelerate progenitor cell levels led to a reduced bone marrow response and failure to produce a marked thrombocytosis.

Serum immunoglobulin isotype profile of viable and non viable lymphoid cell chimaeras made with nude athymic lpr (lymphoproliferation) mouse recipients.

C57BL/6 mice (B6) which are homozygous at the nu (nude, athymic) and lpr (lymphoproliferation) locus (B6 nulpr) are short-lived. We showed previously that increased survival could be obtained by grafting lymphoid cells from euthymic lpr-homozygous B6 mice (B6 lpr) mice ([lpr----nulpr] chimaeras), but curiously enough not from normal (B6 wild) mice ([wild----nulpr] chimaeras). Moreover female, but not male, [lpr----nulpr] chimaeras developed spleen and lymph node enlargement. In the present paper the distribution and absolute concentrations of all serum immunoglobulin (Ig) isotypes have been determined in these chimaeras and their controls. All chimaeras displayed whole serum Ig levels higher than those of B6 wild mice, suggesting a successful reconstitution of the athymic recipients by the grafted lymphoid cells, but two types of chimaeras were peculiar. The short-lived [wild----nulpr] chimaeras showed a proportion of IgM as high as ungrafted B6 nulpr mice, suggesting a deficient down-regulation of IgM production by the grafted B6 wild-type lymphoid cells. The [lpr----nulpr] female chimaeras recovered a long lasting overexpression of all Ig isotypes, like B6 lpr mice, while all the other chimaeras showed a transient overexpression only. Since neither lymphadenopathy nor persistent increase of serum Ig levels were observed in [lpr----nu] chimaeras, our data confirmed the need for a genetically lpr host to allow the significant development of the lpr syndrome.

Elevated gene expression of argininosuccinate synthetase in peripheral lymphocytes from systemic lupus erythematosus (SLE) patients.

Argininosuccinate synthetase (ASS) is a rate-limiting enzyme of urea cycle and functions primarily in the liver, whereas ASS activity is hardly detected in normal lymphocytes. In this study, we examined the level of ASS gene expression in peripheral blood lymphocytes (PBL) from human SLE patients by amplification of reverse-transcribed mRNA using the polymerase chain reaction. We have demonstrated that (a) approximately 40% of SLE patients exhibited 2.5 to 5 times higher expression of ASS gene in PBL than those of healthy PBL and (b) the elevation of ASS gene expression of PBL significantly correlates with the active pathogenesis of SLE patients according to the criteria of Japanese Ministry of Health and Welfare (p < 0.001 by student's two-tailed t-test). Thus, it is suggested that ASS gene expression is a promising marker of hyperactivated lymphocytes uniquely generated in patients with systemic autoimmune disease.

Molecular pathology of inherited erythrocyte membrane disorders: hereditary spherocytosis and elliptocytosis.

Hereditary spherocytosis and elliptocytosis are common genetic defects of the red blood cell membrane skeleton. In recent years rapid advances have been made in the knowledge of the protein structure and assembly of the cytoskeleton. Thanks to the wide use of protein analysis methods several alterations have been discovered in functionally important domains of the different cytoskeletal proteins in these diseases. The cloning of cDNA for the majority of the cytoskeletal proteins allows us to begin elucidating some of these defects at the DNA level. This paper will review the effects of recent advances upon: cytoskeleton structure and assembly; molecular pathology of spherocytosis, elliptocytosis and pyropoikilocytosis.

Pathogenic significance of serum components in the development of autoimmune polyarthritis in MRL/Mp mice bearing the lymphoproliferation gene.

MRL/Mp mice bearing the lymphoproliferation gene (lpr) (MRL/Mp-lpr/lpr) spontaneously develop polyarthritis, associated with autoimmune traits, including rheumatoid factor production, which resembles rheumatoid arthritis. To investigate possible arthritogenic activity of serum of these mice, intraarticular injections of the serum components to knee joints of nonarthritic MRL/Mp mice not bearing the lpr gene (MRL/Mp(-)+/+) were performed. Two fractions from the serum were obtained by a gel chromatography. The void fraction (VF), but not the nonvoid fraction (NVF), induced acute inflammatory lesions in the joints by single injection, and destructive arthritis by repeated injections. VF had immune complex activity, and contained a large amount of cryoglobulin, which in itself was found arthritogenic. These findings indicate that the serum components of MRL/Mp-lpr/lpr mice have a potency to cause destructive arthritis. These results are direct evidence in a syngeneic animal model system, which suggests the pathogenic significance of serum components in rheumatoid arthritis.

Increased macrophage colony-stimulating factor in neonatal and adult autoimmune MRL-lpr mice.

Abnormal macrophages in MRL-lpr mice are implicated in the pathogenesis of autoimmune disease. These mice die of lupus nephritis by 5 to 6 months of age. This study reports that MRL-lpr mice have an increased level of circulating macrophage colony-stimulating factor (M-CSF) detectable as early as 1 week of age. Macrophage colony-stimulating factor decreased between 2 and 4 months and then steadily increased beginning at 4 months of age. In contrast, M-CSF was not detected in sera from congenic MRL-++ mice, normal C3H/FeJ mice, two other mouse strains with the lpr gene (B6-lpr and C3H-lpr), or another lupus model, the NZB/W mouse. These observations indicate that the lpr gene alone is not responsible for inducing this growth factor, and elevated M-CSF is not required for all forms of murine lupus. The entire source of serum M-CSF is not clear. The unique T cells regulated by the lpr gene are not responsible for the increased serum M-CSF levels, as no M-CSFs could be detected in supernatants from cultured lymph nodes from MRL-lpr mice, and the steady-state levels of M-CSF mRNA in lymph nodes and spleens in MRL-lpr, C3H-lpr mice and in their respective congenic strains were similar. The steady-state M-CSF mRNA transcripts in liver, lung, and bone marrow in MRL-lpr, MRL-++, and C3H/FeJ mice were also similar. Macrophage colony-stimulating factor transcripts were clearly elevated in the kidneys of MRL-lpr mice, suggesting a renal source of circulating M-CSF. The increase of M-CSF might be responsible for the increased numbers and enhanced functions of macrophages, which in turn cause tissue destruction in MRL-lpr mice.

Compensatory mechanisms in platelet production: the response of Sl/Sld mice to 5-fluorouracil.

Sl/Sld mice are a unique animal model for studying platelet production in that they sustain normal platelet mass despite reduced marrow activity. The aim of this study was to determine if the compensatory mechanisms operating in these mice could be augmented by further reducing bone marrow activity with the drug 5-fluorouracil (5-FU), known to induce a strong stimulatory effect on platelet production. The platelet recovery in Sl/Sld mice after 5-FU administration contrasted that found in their normal littermates. Sl/Sld mice did not display the sustained thrombocytosis that was observed in +/+ mice between days 10 and 14. Platelet number was elevated in Sl/Sld mice at day 20, when the marrow megakaryocyte compartment had normalized. A significant increase in marrow megakaryocyte number and size was observed at days 8 and 11 in both +/+ and Sl/Sld mice after 5-FU administration. The data suggest that the increase in megakaryocyte size and number following 5-FU treatment was not able to significantly contribute to a sustained rebound thrombocytosis at the time of increased marrow megakaryocytopoiesis. It is concluded that the already compromised marrow of Sl/Sld mice is able to respond to the damage invoked by 5-FU to produce larger than normal megakaryocytes. In contrast to normal mice (+/+ littermates), the increase in marrow megakaryocytopoiesis observed does not lead to a thrombocytosis, indicating that platelet production and release in Sl/Sld mice cannot be further amplified by a strong marrow stimulation.

Analysis of atherosclerosis susceptibility in mice with genetic defects in platelet function.

To determine whether platelets contribute to the development of atherosclerosis, we compared the severity of atherosclerosis in susceptible C57BL/6 mice carrying either a normal or a variant phenotype for platelet function. Five genetically distinct mutants with increased bleeding times and abnormal dense granules were used: maroon (ru-2mr), light ear (le), ruby eye (ru), beige (bg1), and pale ear (ep). After a 14-week consumption of an atherogenic diet, three mutants had significantly less disease involvement than the control: light ear, maroon, and ruby eye. In contrast, pale ear ahd lesions similar to control animals. After 48 weeks, the two mutants with the least degree of atherosclerosis at 14 weeks, light ear and ruby eye, showed greater than 50% survival. In contrast, no animals from the beige, pale ear, or the normal C57BL/6 strains survived. To determine whether a specific biochemical component of platelet function is related to atherosclerosis, we measured serotonin found in dense granules. Serotonin showed no correlation with each mutant's atherosclerosis susceptibility. These results indicate that some particular component of platelet function affects atherosclerosis. That component is intact in pale ear, moderately affected in beige and maroon, and severely affected in light ear and ruby eye. The identity of that component remains an interesting question whose answer may provide further insight into the atherosclerotic disease process.