Defective Reelin/Dab1 signaling pathways associated with disturbed hippocampus development of homozygous yotari mice.

Homozygous Dab1 yotari mutant mice, Dab1yot (yot/yot) mice, have an autosomal recessive mutation of Dab1 and show reeler-like phenotype including histological abnormality of the cerebellum, hippocampus, and cerebral cortex. We here show abnormal hippocampal development of yot/yot mice where granule cells and pyramidal cells fail to form orderly rows but are dispersed diffusely in vague multiplicative layers. Possibly due to the positioning failure of granule cells and pyramidal cells and insufficient synaptogenesis, axons of the granule cells did not extend purposefully to connect with neighboring regions in yot/yot mice. We found that both hippocampal granule cells and pyramidal cells of yot/yot mice expressed proteins reactive with the anti-Dab1 antibody. We found that Y198- phosphorylated Dab1 of yot/yot mice was greatly decreased. Accordingly the downstream molecule, Akt was hardly phosphorylated. Especially, synapse formation was defective and the distribution of neurons was scattered in hippocampus of yot/yot mice. Some of neural cell adhesion molecules and hippocampus associated transcription factors of the neurons were expressed aberrantly, suggesting that the Reelin-Dab1 signaling pathway seemed to be importantly involved in not only neural migration as having been shown previously but also neural maturation and/or synaptogenesis of the mice. It is interesting to clarify whether the defective neural maturation is a direct consequence of the dysfunctional Dab1, or alternatively secondarily due to the Reelin-Dab1 intracellular signaling pathways.

Nephron-deficient Fvb mice develop rapidly progressive renal failure and heavy albuminuria involving excess glomerular GLUT1 and VEGF.

Reduced nephron numbers may predispose to renal failure. We hypothesized that glucose transporters (GLUTs) may contribute to progression of the renal disease, as GLUTs have been implicated in diabetic glomerulosclerosis and hypertensive renal disease with mesangial cell (MC) stretch. The Os (oligosyndactyly) allele that typically reduces nephron number by approximately 50%, was repeatedly backcrossed from ROP (Ra/+ (ragged), Os/+ (oligosyndactyly), and Pt/+ (pintail)) Os/+ mice more than six times into the Fvb mouse background to obtain Os/+ and +/+ mice with the Fvb background for study. Glomerular function, GLUT1, signaling, albumin excretion, and structural and ultrastructural changes were assessed. The FvbROP Os/+ mice (Fvb background) exhibited increased glomerular GLUT1, glucose uptake, VEGF, glomerular hypertrophy, hyperfiltration, extensive podocyte foot process effacement, marked albuminuria, severe extracellular matrix (ECM) protein deposition, and rapidly progressive renal failure leading to their early demise. Glomerular GLUT1 was increased 2.7-fold in the FvbROP Os/+ mice vs controls at 4 weeks of age, and glucose uptake was increased 2.7-fold. These changes were associated with the activation of glomerular PKCbeta1 and NF-kappaB p50 which contribute to ECM accumulation. The cyclic mechanical stretch of MCs in vitro, used as a model for increased MC stretch in vivo, reproduced increased GLUT1 at 48 h, a stimulus for increased VEGF expression which followed at 72 h. VEGF was also shown to act in a positive feedback manner on MC GLUT1, increasing GLUT1 expression, glucose uptake and fibronectin (FN) accumulation in vitro, whereas antisense suppression of GLUT1 largely blocked FN upregulation by VEGF. The FvbROP Os/+ mice exhibited an early increase in glomerular GLUT1 leading to increased glomerular glucose uptake PKCbeta1, and NF-kappaB activation, with excess ECM accumulation. A GLUT1-VEGF-GLUT1 positive feedback loop may play a key role in contributing to renal disease in this model of nondiabetic glomerulosclerosis.

Defective carbohydrate metabolism in mice homozygous for the tubby mutation.

Tub is a member of a small gene family, the tubby-like proteins (TULPs), with predominant expression in neurons. Mice carrying a mutation in Tub develop retinal and cochlear degeneration as well as late-onset obesity with insulin resistance. During behavioral and metabolic testing, we found that homozygous C57BL/6J-Tub(tub) mice have a lower respiratory quotient than C57BL/6J controls before the onset of obesity, indicating that tubby homozygotes fail to activate carbohydrate metabolism and instead rely on fat metabolism for energy needs. In concordance with this, tubby mice show higher excretion of ketone bodies and accumulation of glycogen in the liver. Quantitation of liver mRNA levels shows that, during the transition from light to dark period, tubby mice fail to induce glucose-6-phosphate dehydrogenase (G6pdh), the rate-limiting enzyme in the pentose phosphate pathway that normally supplies NADPH for de novo fatty acid synthesis and glutathione reduction. Reduced G6PDH protein levels and enzymatic activity in tubby mice lead accordingly to lower levels of NADPH and reduced glutathione (GSH), respectively. mRNA levels for the lipolytic enzymes acetyl-CoA synthetase and carnitine palmitoyltransferase are increased during the dark cycle and decreased during the light period, and several citric acid cycle genes are dysregulated in tubby mice. Examination of hypothalamic gene expression showed high levels of preproorexin mRNA leading to accumulation of orexin peptide in the lateral hypothalamus. We hypothesize that abnormal hypothalamic orexin expression leads to changes in liver carbohydrate metabolism and may contribute to the moderate obesity observed in tubby mice.

Fat aussie--a new Alström syndrome mouse showing a critical role for ALMS1 in obesity, diabetes, and spermatogenesis.

Mutations in the human ALMS1 gene are responsible for Alström syndrome, a disorder in which key metabolic and endocrinological features include childhood-onset obesity, metabolic syndrome, and diabetes, as well as infertility. ALMS1 localizes to the basal bodies of cilia and plays a role in intracellular trafficking, but the biological functions of ALMS1 and how these relate to the pathogenesis of obesity, diabetes, and infertility remain unclear. Here we describe a new mouse model of Alström syndrome, fat aussie, caused by a spontaneous mutation in the Alms1 gene. Fat aussie (Alms1 foz/foz) mice are of normal weight when young but, by 120 d of age, they become obese and hyperinsulinemic. Diabetes develops in Alms1 foz/foz mice accompanied by pancreatic islet hyperplasia and islet cysts. Female mice are fertile before the onset of obesity and metabolic syndrome; however, male fat aussie mice are sterile due to a progressive germ cell loss followed by an almost complete block of development at the round-to-elongating spermatid stage of spermatogenesis. In conclusion, Alms1 foz/foz mouse is a new animal model in which to study the pathogenesis of the metabolic and fertility defects of Alström syndrome, including the role of ALMS1 in appetite regulation, pathogenesis of the metabolic syndrome, pancreatic islet physiology, and spermatogenesis.

Raf-1 sets the threshold of Fas sensitivity by modulating Rok-alpha signaling.

Ablation of the Raf-1 protein causes fetal liver apoptosis, embryonic lethality, and selective hypersensitivity to Fas-induced cell death. Furthermore, Raf-1-deficient cells show defective migration as a result of the deregulation of the Rho effector kinase Rok-alpha. In this study, we show that the kinase-independent modulation of Rok-alpha signaling is also the basis of the antiapoptotic function of Raf-1. Fas activation stimulates the formation of Raf-1-Rok-alpha complexes, and Rok-alpha signaling is up-regulated in Raf-1-deficient cells. This leads to increased clustering and membrane expression of Fas, which is rescued both by kinase-dead Raf-1 and by interfering with Rok-alpha or its substrate ezrin. Increased Fas clustering and membrane expression are also evident in the livers of Raf-1-deficient embryos, and genetically reducing Fas expression counteracts fetal liver apoptosis, embryonic lethality, and the apoptotic defects of embryonic fibroblasts. Thus, Raf-1 has an essential function in regulating Fas expression and setting the threshold of Fas sensitivity during embryonic life.

Mouse model of male germ cell apoptosis in response to a lack of hormonal stimulation.

As a prerequisite for studies using mutant mice, we established a mouse model for induction of male germ cell apoptosis after deprivation of gonadotropins and intratesticular testosterone (T). We employed a potent long acting gonadotropin-releasing hormone antagonist (GnRH-A), acyline, alone or in combination with an antiandrogen, flutamide for effective induction of germ cell apoptosis in mice. Combined treatment with continuous release of acyline (3 mg/kg BW/day) with flutamide (in the form of sc pellets of 25 mg) resulted in almost the same level of suppression of spermatogenesis, as judged by testis weight and by germ cell apoptotic index, in 2 weeks as that reported for rats after treatment with 1.25 mg/kg BW Nal-Glu GnRH-A for the same time period. Within the study paradigm, the maximum suppression of spermatogenesis occurred after a single sc injection of high (20 mg/kg BW) dose of acyline with flutamide. The combined treatment resulted in complete absence of elongated spermatids. Germ cell counts at stages VII-VIII showed a significant (P < 0.05) reduction in the number of preleptotene (27.1%) and pachytene spermatocytes (81.9%), and round spermatids (96.6%) in acyline + flutamide group in comparison with controls. In fact, treatment with a single high (20 mg/kg BW) dose of acyline combined with flutamide in mice achieved same or greater level of suppression, measured by germ cell counts at stages VII-VIII, in two weeks when compared with those reported after daily treatment with Nal-Glu GnRH-A for 4 weeks in rats. Both plasma and testicular T levels were markedly suppressed after administration of acyline alone either by miniosmotic pump or by a single sc injection. Addition of flutamide to acyline had no discernible effect on plasma or intratesticular T levels when compared with acyline alone. These results demonstrate that optimum suppression of spermatogenesis through increased germ cell death is only possible in mice if total abolition of androgen action is achieved and further emphasize the usefulness of acyline + flutamide treated mice as a suitable model system to study hormonal regulation of testicular germ cell apoptosis.

Sex differences in expression of transforming growth factor-alpha and epidermal growth factor receptor mRNA in Waved-1 and C57Bl6 mice.

A reduction of transforming growth factor-alpha (TGFalpha) expression in the spontaneous Waved-1 (Wa-1) mutant mouse causes specific behavioral and anatomical changes, including reduced fear learning and stress response and enlarged lateral ventricles. These alterations are observed predominantly in male Wa-1 mice after puberty. We hypothesized that regional differences in the expression of TGFalpha and its receptor, epidermal growth factor receptor (EGFR), may regulate the sexual dimorphism of the brain structures and functions during postnatal development. In general, fear learning-associated structures, including hippocampus and amygdala, showed maximum expression before puberty, regardless of genotype. In contrast, an overall temporal delay in the rise of both transcript levels, which peaked around or after puberty onset, was observed for the major stress regulatory hypothalamo-pituitary-adrenal axis. This pattern of expression was reversed for amygdala EGFR and hypothalamus TGFalpha and EGFR transcripts in males. When regional TGFalpha expression was compared between control and Wa-1 mice, far more complex patterns than expected were observed that revealed sex- and structure-dependent differences. In fact, the amygdala, hypothalamus, and pituitary TGFalpha expression pattern in Wa-1 exhibited a clear sex dependency across various age groups. Surprisingly, there was no compensatory up-regulation of the EGFR transcript in Wa-1 mice. The observed expression patterns of the TGFalpha signaling system during normal development and in the Wa-1 mutant mouse suggest complex sex- and age-dependent transcription regulatory mechanisms.

Development of the inner ear in Splotch mutant mice.

The Splotch mouse, a Pax 3 mutation, represents a model of Waardenburg syndrome I. We show that the homozygous Splotch mutation (Sp(2H)) is associated with severe defects that prevent the formation of the cochlea and vestibulo-cochlear ganglion. To clarify the role of Pax 3 in inner ear formation, we examined the expression of polysialic acid (PSA) associated with neural cell adhesion molecule (NCAM). In accordance with the occurrence of phenotypic abnormalities, PSA NCAM was expressed early in otocyst development in the otic epithelium and the vestibulo-cochlear anlage. During the period of vestibular and cochlear ganglia formation, PSA NCAM expression was decreased. In the late phase of embryonic development, the expression of calcium binding proteins (S100) in the vestibulo-cochlear ganglion was also decreased. Minor differences in S100 immunostaining were found postnatally between the cochleas of heterozygous and wild type animals.

Physiological functions of Pten in mouse tissues.

PTEN is a tumor suppressor gene mutated in many human sporadic cancers and in hereditary cancer syndromes such as Cowden disease, Bannayan-Zonana syndrome and Lhermitte-Duclos disease. The major substrate of PTEN is PIP3, a second messenger molecule produced following PI3K activation induced by variety of stimuli. PIP3 activates the serine-threonine kinase PKB/Akt which is involved in anti-apoptosis, proliferation and oncogenesis. In mice, heterozygosity for a null mutation of Pten (Pten(+/-) mice) frequently leads to the development of a variety of cancers and autoimmune disease. Homozygosity for the null mutation (Pten (-/-) mice) results in early embryonic lethality, precluding the functional analysis of Pten in various organs. To investigate the physiological functions of Pten in viable mice, various tissue-specific Pten mutations have been generated using the Cre-loxP system. This review will summarize the phenotypes of conditional mutant mice lacking Pten function in specific tissues, and discuss how these phenotypes relate to the physiological roles of Pten in various organ systems.

Characterization of properties underlying rhythmicity in mouse portal vein.

We have used sharp intracellular and patch clamp electrophysiology, together with mechanical recordings and immunohistochemistry to characterize some of the properties underlying spontaneous rhythmicity in isolated murine portal vein. Mechanical recordings revealed that isolated whole portal veins were spontaneously active and generated regular contractions every 5-15-s that persisted in the presence of cyclopiazonic acid (CPA) (10 microM) or thapsigargin (100 nM). Intracellular recordings from smooth muscle cells revealed spontaneous depolarizations (SDs) in membrane potential, which were abolished by nifedipine (1 microM). Whole cell patch clamp recordings from isolated smooth muscle cells revealed an inward "pacemaker" current (I(H)) at negative potentials. Immunohistochemical studies failed to detect the presence of Kit-immunoreactive cells in portal veins of wild type mice, but were consistently observed in the small intestine. Furthermore, portal veins obtained from W/W(v) mutant mice, which lack full expression of the tyrosine-kinase, c-Kit, were also rhythmically active and were not different from wild type mice, in either their electrical or mechanical properties. These results show that both the wild type and W/W(v) mutant mouse portal vein are rhythmically active in vitro. However, pacemaker activity in this blood vessel occurs in the absence of Kit-immunoreactive cells; and is not critically dependent upon release of Ca(2+) from intracellular stores. The rhythmic pacemaker activity of mouse portal vein does involve L-type Ca(2+) currents, and possibly pacemaker conductances intrinsic to the smooth muscle.

FGF signalling is required for differentiation-induced cytoskeletal reorganisation and formation of actin-based processes by podocytes.

To examine the potential role of fibroblast growth factor (FGF) signalling during cell differentiation, we used conditionally immortalised podocyte cells isolated from kidneys of Fgf2 mutant and wild-type mice. Wild-type mouse podocyte cells upregulate FGF2 expression when differentiating in culture, as do maturing podocytes in vivo. Differentiating wild-type mouse podocyte cells undergo an epithelial to mesenchymal-like transition, reorganise their actin cytoskeleton and extend actin-based cellular processes; all of these activities are similar to the activity of podocytes in vivo. Molecular analysis of Fgf2 mutant mouse podocyte cells reveals a general disruption of FGF signalling as expression of Fgf7 and Fgf10 are also downregulated. These FGF mutant mouse podocyte cells in culture fail to activate mesenchymal markers and their post-mitotic differentiation is blocked. Furthermore, mutant mouse podocyte cells in culture fail to reorganise their actin cytoskeleton and form actin-based cellular processes. These studies show that FGF signalling is required by cultured podocytes to undergo the epithelial to mesenchymal-like changes necessary for terminal differentiation. Together with other studies, these results point to a general role for FGF signalling in regulating cell differentiation and formation of actin-based cellular processes during morphogenesis.

The mouse osteopetrotic grey-lethal mutation induces a defect in osteoclast maturation/function.

The osteopetrotic grey-lethal (gl) mouse mutant displays many similarities to the human malignant autosomal-recessive form of osteopetrosis. In this study, we show that the gl osteopetrotic bone phenotype is characterized by the presence of numerous differentiated multinucleated osteoclasts. A significant increase in the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts was detected in vivo, suggesting induction of differentiation in the osteoclast lineage as a compensatory mechanism. These gl osteoclast cells demonstrated a defective cytoskeletal reorganization and an underdeveloped ruffled border, a membrane structure essential for active bone resorption. Accordingly, resorption activity of these cells is markedly impaired by four- to tenfold as evaluated with the pit formation assay. This low bone resorption in gl osteoclasts is highly reminiscent of the loss in key enzymes, V-ATPase or cathepsin-K, and in signaling factors, Src or TRAF-6, which were shown not to be significantly altered in gl osteoclasts. Thus, independently of a deficiency in V-ATPase, Src, cathepsin-K, and TRAF-6, the gl mutation results in increased number of osteoclasts, characterized by a disrupted cytoskeleton and an underdeveloped ruffled border.

A soluble form of the murine common gamma chain is present at high concentrations in vivo and suppresses cytokine signaling.

The common gamma-chain (gammac) is a component of the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 and is essential for their signal transduction. Western blotting and a newly established enzyme-linked immunosorbent assay detected substantial constitutive levels (50-250 ng/mL) of soluble gammac (sgammac) in sera of murine inbred strains. It was demonstrated that purified immune cells, such as T, B, and natural killer cells, and macrophages released this protein after activation. Transfection experiments with cDNA encoding the full-length gammac showed that shedding of the transmembrane receptor led to the release of sgammac. The shedding enzymes, however, appeared to be distinct from those cleaving other cytokine receptors because inhibitors of metalloproteases (eg, TAPI) did not influence sgammac release. In vivo, superantigen-induced stimulation of T cells enhanced sgammac serum concentrations up to 10-fold within 6 hours. Because these findings demonstrated regulated expression of a yet unknown molecule in the immune response, further experiments were performed to assess the possible function(s) of sgammac. A physiological role of sgammac was indicated by its capacity to specifically inhibit cell growth induced by gammac-dependent cytokines. Mutational analysis revealed that the C-terminus and the WSKWS motif are essential for the cytokine inhibitory effect of the sgammac and for binding of the molecule to cytokine receptor-expressing cells. Thus, competitive displacement of the transmembrane gammac by excess sgammac is the most likely mechanism of cell growth inhibition. It was implied that naturally produced sgammac is a negative modulator of gammac-dependent cytokines.

Loss of normal profilaggrin and filaggrin in flaky tail (ft/ft) mice: an animal model for the filaggrin-deficient skin disease ichthyosis vulgaris.

Flaky tail (gene symbol ft) is an autosomal recessive mutation in mice that results in a dry, flaky skin, and annular tail and paw constrictions in the neonatal period. Previous studies demonstrated that the ft mutation maps to the central region of mouse chromosome 3, in the vicinity of the epidermal differentiation complex, a gene locus that includes many nonkeratin genes expressed in epidermis. In this study we report a detailed characterization of the flaky tail mouse. Affected homozygous ft/ft mice exhibit large, disorganized scales on tail and paw skin, marked attenuation of the epidermal granular layer, mild acanthosis, and orthokeratotic hyperkeratosis. Biochemical analysis demonstrated that ft/ft mice lacked normal high molecular profilaggrin (approximately 500 kDa), and instead expressed a lower molecular weight form of profilaggrin (220 kDa) that is not proteolytically processed to profilaggrin intermediates or filaggrin. Mutant mice lacked the large, irregular F-type keratohyalin granules that contain profilaggrin, and filaggrin was absent from the cornified layers of ft/ft epidermis. The expression of epidermal keratins was unchanged, whereas the cornified envelope proteins involucrin and loricrin were increased in ft/ft epidermis. Cultured ft/ft keratinocytes also synthesized reduced amounts of profilaggrin mRNA and protein, demonstrating that the defect in profilaggrin expression is intrinsic to epidermal cells. These findings demonstrate that flaky tail mice express an abnormal profilaggrin polypeptide that does not form normal keratohyalin F-granules and is not proteolytically processed to filaggrin. We propose that the absence of filaggrin, and in particular the hygroscopic, filaggrin-derived amino acids that are thought to function in epidermal hydration, underlies the dry, scaly skin characteristic of ft/ft mice. This animal model provides a tool for understanding the role of filaggrin in normal epidermal function and may provide insight into the molecular basis of the filaggrin-deficient human skin disorder ichthyosis vulgaris. J Invest Dermatol 115:1072-1081 2000

Genetic defects in postsqualene cholesterol biosynthesis.

In humans and mice, four different genetic defects in the nine biosynthetic steps from lanosterol to cholesterol have been identified. They impair the activity of a putative C3-sterol dehydrogenase (Nshdl, X-linked dominant bare patches/striated mutation in mice), the sterol delta 8-delta 7 isomerase/EBP (Ebp, X-linked dominant tattered mutation in mice; chondrodysplasia punctata (CDPX2) in humans), the delta 24-sterol reductase (autosomal recessive desmosterolosis) and the delta 7-sterol reductase (DHCR7 gene, autosomal recessive Smith-Lemli-Opitz syndrome in humans). These inborn errors in postsqualene cholesterol metabolism result in dysmorphogenetic syndromes of variable severity. The X-linked dominant mutations result in mosaicism in females, as a result of X-inactivation, and midgestational lethality in males. The mechanisms by which the depletion of cholesterol or the accumulation of intermediates impair morphogenetic programs are unclear. So far, no cellular processes that require an intact cholesterol biosynthetic pathway have been identified, although the morphogenetic hedgehog-patched signaling cascade is a candidate.

P47(phox)-deficient NADPH oxidase defect in neutrophils of diabetic mouse strains, C57BL/6J-m db/db and db/+.

Deficiencies in neutrophil NADPH oxidase proteins have been demonstrated in humans with chronic granulomatous disease. However, no spontaneous mutation in murine NADPH oxidase has been reported. In this study we report that neutrophils from the diabetic mouse strains, C57BL/6J-m heterozygous lean (lepr(db/+)) and homozygous obese (lepr(db/db)) mice produced no superoxide on stimulation. An absence of intact p47(phox) but not other oxidase proteins was observed in both mouse strains through the use of immunoblotting. Molecular analysis by reverse transcriptase-polymerase chain reaction identified three abnormal p47phox mRNA transcripts. Sequencing of genomic DNA of p47(phox) revealed a point mutation at the -2 position of exon 8, which is consistent with aberrant splicing of the p47(phox) transcript. These results indicate that the C57BL/6J-m db/db and db/+ mice are the first spontaneously derived murine model of NADPH oxidase deficiency involving a p47(phox) mutation.

Chemoprevention of spontaneous intestinal adenomas in the adenomatous polyposis coli Min mouse model with aspirin.

BACKGROUND & AIMS: Colorectal cancer is a significant source of morbidity and mortality in the United States and other Western countries. Epidemiological and experimental data indicate that regular use of aspirin reduces colon cancer risk. This study was designed to determine if aspirin would significantly inhibit gastrointestinal tumor formation in a mouse model of familial adenomatous polyposis. METHODS: Six-week-old male and female C57BL/6J +/+ (control) and C57BL/6J ApcMin/+ (Min) mice were fed either a control AIN-76A diet or one supplemented with 250 or 500 parts per million (ppm) aspirin (n = 6 per group) for 7 weeks. RESULTS: All of the Min mice, but no control mice, developed gastrointestinal tumors. Aspirin significantly reduced tumor multiplicity (number of tumors per mouse) in the small intestine, but not the colon, from an average of 35.8 tumors per mouse (control diet) to 16 and 18.5 tumors per mouse with 250 and 500 ppm aspirin, respectively. Total tumor load (sum of tumor diameters per mouse) was also significantly reduced, from 93.2 mm in total diameter to 40. 4 and 45.0 mm with 250 and 500 ppm aspirin, respectively. Results were not significantly different because of sex or aspirin dose. CONCLUSIONS: High doses of aspirin are effective chemopreventive agents in a mouse model of spontaneous intestinal tumor formation.

Expression of the mouse Gli and Ptc genes is adjacent to embryonic sources of hedgehog signals suggesting a conservation of pathways between flies and mice.

The three mouse Gli genes are putative transcription factors which are the homologs of cubitus interruptus (ci) in Drosophila. Along with the gene patched (Ptc), ci has been implicated in the hedgehog (Hh) signal transduction pathway. To assess the role of Gli in embryogenesis, we compared its expression with that of Ptc and Hh family members in mouse. We found that Gli and Ptc are expressed in similar domains in diverse regions of the developing mouse embryo and these regions are adjacent to Hh signals. We also show that Gli is expressed ectopically along with Ptc and Shh in Strong's luxoid mutant mice. These results are consistent with conservation of the Hh signal transduction pathway in mice with Gli potentially mediating Hh signaling in multiple regions of the developing embryo.

Distribution of injected iron 59 and manganese 54 in hypotransferrinemic mice.

Transferrin has been proposed as the mobilization protein for iron and manganese. To better understand the role of transferrin in the transport of these metals, we studied the tissue distribution of injected iron 59 and manganese 54 in the hypotransferrinemic (Hp) mouse mutant. The Hp mouse has a mutation in the transferrin gene and produces < 1% of normal transferrin levels. The tissue distribution of 59Fe and 54Mn in Hp mice was compared with that in animals heterozygous for the Hp mutation (50% transferrin levels) and wild-type animals. Formed elements in the brain, liver, spleen, heart, sternum/rib, plasma, and blood were analyzed for isotope incorporation at 24 hours, 7 days, and 4 weeks after injection. Tissue distribution of both 59Fe and 54Mn was similar in wild-type and heterozygote animals, indicating that decreased transferrin concentration and increased saturation did not influence the tissue distribution of the injected metals. The absence of transferrin in the Hp mutant was associated with abnormal tissue distribution of radiolabeled iron; there was 4 times more 59Fe than normal in the Hp liver and 10 times less 59Fe in the spleen and blood formed elements than normal. Injected manganese also accumulated at abnormally high levels in the Hp mouse liver. Distribution of either metal to the brain, heart, and sternum/rib was not affected by the absence of plasma transferrin. These results reveal that transferrin is required for proper targeting of manganese and iron, especially from the liver to other organs, but further indicate that nontransferrin transport mechanisms for iron and manganese must exist.

Impairment of the nitric oxide/cyclic GMP pathway in cerebellar slices prepared from the hph-1 mouse.

In this study, the effect of tetrahydrobiopterin deficiency on the nitric oxide/cGMP pathway has been investigated in cerebellar slices derived from the hph-1 mouse. This animal displays a partial deficiency of tetrahydrobiopterin. Basal levels of cGMP were significantly reduced (-29.5%) in the hph-1 mouse cerebellum compared to controls. Following kainate stimulation (500 microM) cGMP levels increased in both control and hph-1 preparations but were again significantly lower (-29.1%) in the hph-1 mouse. Exposure of slices to the nitric oxide donors, S-nitroso-N-acetylpenicillamine and S-nitroso-glutathione, revealed no difference in cGMP accumulation between the two groups. These findings suggest that the cerebellar nitric oxide/cGMP pathway may be impaired in partial tetrahydrobiopterin deficiency states due to diminished nitric oxide formation.