Search

Tetrahydrobiopterin (BH4) is an essential cofactor for the aromatic amino acid hydroxylases, alkylglycerol monooxygenase, and nitric oxide synthases (NOS). Inborn errors of BH4 metabolism lead to severe insufficiency of brain monoamine neurotransmitters while augmentation of BH4 by supplementation or stimulation of its biosynthesis is thought to ameliorate endothelial NOS (eNOS) dysfunction, to protect from (cardio-) vascular disease and/or prevent obesity and development of the metabolic syndrome. We have previously reported that homozygous knock-out mice for the 6-pyruvolytetrahydropterin synthase (PTPS; Pts-ko/ko) mice with no BH4 biosynthesis die after birth. Here we generated a Pts-knock-in (Pts-ki) allele expressing the murine PTPS-p.Arg15Cys with low residual activity (15% of wild-type in vitro) and investigated homozygous (Pts-ki/ki) and compound heterozygous (Pts-ki/ko) mutants. All mice showed normal viability and depending on the severity of the Pts alleles exhibited up to 90% reduction of PTPS activity concomitant with neopterin elevation and mild reduction of total biopterin while blood L-phenylalanine and brain monoamine neurotransmitters were unaffected. Yet, adult mutant mice with compromised PTPS activity (i.e., Pts-ki/ko, Pts-ki/ki or Pts-ko/wt) had increased body weight and elevated intra-abdominal fat. Comprehensive phenotyping of Pts-ki/ki mice revealed alterations in energy metabolism with proportionally higher fat content but lower lean mass, and increased blood glucose and cholesterol. Transcriptome analysis indicated changes in glucose and lipid metabolism. Furthermore, differentially expressed genes associated with obesity, weight loss, hepatic steatosis, and insulin sensitivity were consistent with the observed phenotypic alterations. We conclude that reduced PTPS activity concomitant with mildly compromised BH4-biosynthesis leads to abnormal body fat distribution and abdominal obesity at least in mice. This study associates a novel single gene mutation with monogenic forms of obesity.

Tyrosine hydroxylase catalyses the hydroxylation of L-tyrosine to l-DOPA, the rate-limiting step in the synthesis of catecholamines. Mutations in the TH gene encoding tyrosine hydroxylase are associated with the autosomal recessive disorder tyrosine hydroxylase deficiency, which manifests phenotypes varying from infantile parkinsonism and DOPA-responsive dystonia, also termed type A, to complex encephalopathy with perinatal onset, termed type B. We generated homozygous Th knock-in mice with the mutation Th-p.R203H, equivalent to the most recurrent human mutation associated with type B tyrosine hydroxylase deficiency (TH-p.R233H), often unresponsive to l-DOPA treatment. The Th knock-in mice showed normal survival and food intake, but hypotension, hypokinesia, reduced motor coordination, wide-based gate and catalepsy. This phenotype was associated with a gradual loss of central catecholamines and the serious manifestations of motor impairment presented diurnal fluctuation but did not improve with standard l-DOPA treatment. The mutant tyrosine hydroxylase enzyme was unstable and exhibited deficient stabilization by catecholamines, leading to decline of brain tyrosine hydroxylase-immunoreactivity in the Th knock-in mice. In fact the substantia nigra presented an almost normal level of mutant tyrosine hydroxylase protein but distinct absence of the enzyme was observed in the striatum, indicating a mutation-associated mislocalization of tyrosine hydroxylase in the nigrostriatal pathway. This hypomorphic mouse model thus provides understanding on pathomechanisms in type B tyrosine hydroxylase deficiency and a platform for the evaluation of novel therapeutics for movement disorders with loss of dopaminergic input to the striatum., (© The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Splicing is an essential cellular process to generate mature transcripts from pre-mRNA. It requires the splice factor U1 small nuclear ribonucleoprotein (U1), which promotes exon recognition by base-pairing interaction with the splice donor site (SD). After U1 dissociation, exon recognition is maintained by U6 small nuclear ribonucleoproteins (U6). It has been shown that SD mutations lower the binding affinity of U1 and cause splice defects in about 10% of patients with monogenetic diseases. U1 isoforms specifically designed to bind the mutated SD with increased affinity can correct these splice defects. We investigated the applicability of this gene therapeutic approach for different mutated SD positions. A minigene-based splicing assay was established to study a typical SD derived from the gene BBS1. We found that mutations at seven SD positions caused splice defects. In four cases, mutation-adapted U1 isoforms completely corrected these splice defects. Partial correction was found for splice defects induced by the mutation at SD position +5. The limited therapeutic efficacy at this position was alleviated by applying a combined treatment with mutation-adapted U1 and U6. The sequence complementarity between U6 and three SD positions (+4, +5,and +6) was relevant for the outcome of the therapy. Between 30 and 100% of the normal transcripts can be restored. The treatment significantly decreased both exon skipping and intron retention. Massive missplicing of off-target transcripts was not detected. Our study helps to assess the therapeutic efficacy of mutation-adapted U snRNAs in gene therapy and illustrates their strong potential to correct splice defects, which cause many different inherited conditions.

The role of different extracellular matrix (ECM)-degrading enzymes in the normal functioning of the placenta is well documented. Heparan sulphate proteoglycan (HSPG) is an integral constituent of the placental and decidual ECM. Because this proteoglycan specifically interacts with various macromolecules in the ECM, its degradation may disassemble the matrix. Hence, in the case of the placenta, this may facilitate normal placentation and trophoblast invasion. Crude placental specimens were collected from first and third trimester placentas. Heparanase (endo-beta-glucuronidase) was isolated and purified by ammonium sulphate precipitation followed by sequential chromatographies on carboxymethyl-, heparin- and ConA-Sepharose columns. The placental enzyme was further characterized for its molecular weight and specific inhibition by heparin, and was shown to resemble heparanase expressed by highly metastatic tumor cells and activated cells of the immune system. In order to locate the source of heparanase activity in the placenta, primary cytotrophoblast cultures were established. Intact cells, as well as conditioned medium and cell lysates, were analysed for heparanase activity using metabolically sulphate-labelled ECM as a natural substrate. Heparanase was highly active in lysates of cytotrophoblasts. This activity was also expressed by intact cytotrophoblasts seeded on ECM, but no activity could be detected in the culture medium. Incubation of the cytotrophoblasts in contact with ECM resulted in release of ECM-bound basic fibroblast growth factor (bFGF). We propose that the cytotrophoblastic heparanase facilitates placentation, through cytotrophoblast extravasation and localized neovascularization.

Zinc(II) accumulated by platelets has profound effects on platelet activity. This study is focused on the distribution of Zn(II) between human platelet subcellular compartments. After incubation with 86Rb+ and platelet lysis, the organelles were separated by sucrose density gradient centrifugation. Fibrinogen served as a marker for alpha-granules. 86Rb+ and factor XIII served as markers for the cytoplasmic fractions. Zn(II) was found to be distributed between the cytoplasm and the alpha-granules, with variations between different individual units. The total platelet Zn concentration and its relative subcellular distribution were dependent on its extracellular level. Incubation of platelets with 100 microM Zn(II) resulted in a twofold increase of its level in the cytoplasm and by one order of magnitude in the alpha-granules. In addition to the anticipated factor XIII activity in the cytoplasmic pool fraction, we found thrombin-inducible factor XIII activity within the alpha-granules. Immunoblotting confirmed the presence of both the a and b subunits of plasma factor XIII (a2b2 form) in the alpha-granules. As fibrinogen is not synthesized in the platelet, we propose that by virtue of their mutual binding, fibrinogen, Zn(II) and plasma factor XIII-a2b2 are simultaneously taken up into the alpha-granules by endocytosis, presumably through the vehicle of the GPIIb/IIIa fibrinogen receptor. A rationale for co-packaging these components within the alpha-granules is that Zn(II) inhibits factor XIII activity and thereby prevents the premature cross-linking of the concentrated fibrinogen prior to platelet activation and secretion. By contrast, cytoplasmic Zn(II) may increase platelet responsiveness to agonists due to its interaction with cytoplasmic modulators of platelet activity.

Incubation of plasminogen with the subendothelial extracellular matrix (ECM) synthesized by cultured bovine corneal and aortic endothelial cells resulted in generation of fibrinolytic activity, indicated by proteolysis of 125I-fibrin in a time- and dose-dependent manner. Both tissue-type plasminogen activator (t-PA) and urokinase-type plasminogen activator (u-PA) were identified in the ECM by fibrin zymography, immunoblotting, and inhibition of plasminogen activation by anti-u-PA and anti-t-PA antibodies. Most of the ECM-resident plasminogen activator (PA) activity did not originate from intracellular PA release occurring when the endothelial cells were lyzed and the ECM exposed, since a comparable amount of PA was associated with the ECM when the cells were lyzed with Triton X-100 or removed intact by treatment with 2 M urea. Active u-PA and t-PA were released from ECM by treatment with heparanase (endo-beta-D-glucuronidase), indicating that some of the ECM-resident PA activity is sequestered by heparan sulfate side chains. These results indicate that both u-PA and t-PA produced by endothelial cells are firmly sequestered in an active form by the subendothelial ECM. It is suggested that ECM-resident plasminogen activators participate in sequential matrix degradation during cell invasion and tumor metastasis. PA activity may also function in release of ECM-bound growth factors (i.e., basic fibroblast growth factor) and activation of proenzymes (i.e., prothrombin), resulting in modulation of the ECM growth-promoting and thrombogenic properties.

We have characterized the importance of size, sulfation, and anticoagulant activity of heparin in release of basic fibroblast growth factor (bFGF) from the subendothelial extracellular matrix (ECM) and the luminal surface of the vascular endothelium. For this purpose, 125I-bFGF was first incubated with ECM and confluent endothelial cell cultures, or administered as a bolus into the blood of rats, the immobilized 125I-bFGF was then subjected to release by various chemically modified species of heparin and size-homogeneous oligosaccharides derived from depolymerized heparin. Both totally desulfated and N-desulfated heparin failed to release the ECM-bound bFGF. Likewise, substitution of N-sulfate groups of heparin and low molecular weight heparin (fragmin) by acetyl or hexanoyl residues resulted in an almost complete inhibition of bFGF release by these polysaccharides. The presence of O-sulfate groups in heparin increased but was not critical for release of ECM-bound bFGF. Similar structural requirements were identified for release of 125I-bFGF bound to low-affinity sites on the surface of vascular endothelial cells. Oligosaccharides derived from depolymerized heparin and containing as little as 8-10 sugar units were, on a weight basis, equivalent to whole heparin in their ability to release bFGF from ECM. Low-sulfate oligosaccharides were less effective releasers of bFGF as compared to medium- and high-sulfate fractions of the same size oligosaccharides. Heparin fractions with high and low affinity to antithrombin III exhibited a similar high bFGF-releasing activity despite a 200-fold difference in their anticoagulant activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Despite the ubiquitous presence of basic fibroblast growth factor (bFGF) in normal tissues, endothelial cell proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Immunohistochemical staining revealed the localization of bFGF in basement membranes of diverse tissues, suggesting that the extracellular matrix (ECM) may serve as a reservoir for bFGF. Moreover, functional studies indicated that bFGF is an ECM component required for supporting endothelial cell proliferation and neuronal differentiation. We have found that bFGF is bound to heparan sulfate (HS) in the ECM and is released in an active form when the ECM-HS is degraded by heparanase expressed by normal and malignant cells (i.e. platelets, neutrophils, lymphoma cells). It is proposed that restriction of bFGF bioavailability by binding to ECM and local regulation of its release provide a novel mechanism for neovascularization in normal and pathological situations. The subendothelial ECM contains also tissue type- and urokinase type-plasminogen activators which participate in cell invasion and tissue remodeling. These results and studies on the properties of other ECM-immobilized enzymes (i.e. thrombin, plasmin, lipoprotein lipase) and growth factors (GM-CSF, IL-3, osteogenin), suggest that the ECM provides a storage depot for biologically active molecules which are thereby stabilized and protected. This may allow a more localized and persistent mode of action, as compared to the same molecules in a fluid phase.

Neoplastic cells require an appropriate pericellular environment and new formation of stroma and blood vessels in order to constitute a solid tumor. Tumor progression also involves degradation of various extracellular matrix (ECM) constituents. In this review we have focused on the possible involvement of ECM-resident growth factors and enzymes in neovascularization and cell invasion. We demonstrate that the pluripotent angiogenic factor, basic fibroblast growth factor (bFGF) is an ECM component required for supporting cell proliferation and differentiation. Basic FGF has been identified in the subendothelial ECM produced in vitro and in basement membranes of the cornea and blood vessels in vivo. Despite the ubiquitous presence of bFGF in normal tissues, endothelial cell (EC) proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Our results indicate that bFGF is bound to heparan sulfate (HS) in the ECM and is released in an active form when the ECM-HS is degraded by cellular heparanase. We propose that restriction of bFGF bioavailability by binding to ECM and local regulation of its release, provides a novel mechanism for regulation of capillary blood vessel growth in normal and pathological situations. Heparanase activity correlates with the metastatic potential of various tumor cells and heparanase inhibiting molecules markedly reduce the incidence of lung metastasis in experimental animals. Heparanase may therefore participate in both tumor cell invasion and angiogenesis through degradation of the ECM-HS and mobilization of ECM-resident EC growth factors. The subendothelial ECM contains also tissue type- and urokinase type- plasminogen activators (PA), as well as PA inhibitor which may regulate cell invasion and tissue remodeling. Heparanase and the ECM-resident PA participate synergistically in sequential degradation of HS-proteoglycans in the ECM. These results together with similar observations on the properties of other ECM-immobilized enzymes and growth factors, suggest that the ECM provides a storage depot for biologically active molecules which are thereby stabilized and protected. This may allow a more localized, regulated and persistent mode of action, as compared to the same molecules in a fluid phase.

To determine whether there are structural differences between the binding sites for placental lactogen (PL) and GH, we have compared the molecular weights of complexes formed by the covalent cross-linking of [125I]ovine (o) PL and [125I]oGH to hepatic membranes from fetal and pregnant sheep in mid- and late gestation and from postnatal nonpregnant sheep at 3 days to 7 months of age. Specific [125I]oPL binding sites in fetal liver were detected as early as midgestation, and cross-linking of [125I]oPL to fetal hepatic membranes yielded a major radiographic band with a mol wt of 60 +/- 5 K (mean +/- SD). Unlabeled oPL at low concentrations (0.9-9 nM) specifically competed with [125I]oPL for binding to the 60 K complex. In contrast, oGH and oPRL competed for binding to the 60 K complex only at much higher concentrations (greater than or equal to 90 nM). In addition, no specific cross-linking of [125I]oGH or [125I]oPRL to fetal hepatic membranes was observed. These findings suggest the presence of a distinct and unique PL binding site in ovine fetal liver. Since the mol wt of oPL is 22 K, the estimated mol wt of the oPL receptor protein is 38 +/- 5 K. During the first week after birth, there was a striking increase in the number of [125I]oGH binding sites. Cross-linking of [125I]oGH to postnatal liver yielded radiographic bands with apparent mol wts of 75 K and 140 K. The relative potencies of oPL, oGH, and oPRL in competing for binding to the 75 K and 140 K complexes were similar to the relative potencies of these hormones in competing for [125I]oGH binding sites in postnatal liver, suggesting that the 75 K and 140 K bands represent subunits of the oGH receptor bound covalently to [125I]oGH. Cross-linking of [125I]oPL to pregnant and postnatal nonpregnant liver yielded three radiographic bands with mol wts of 60 K, 75 K, and 140 K. The intensities of all three bands were reduced by low concentrations (0.9-9 nM) of oPL. Higher concentrations of oGH abolished the 75 K and 140 K bands but reduced the intensity of the 60 K band by only 20-30%. oPRL had minimal effect on band intensities. These observations suggest the presence of two functionally and structurally distinct receptors in pregnant liver: the oPL receptor, which has high affinity for oPL and low affinity for oGH and oPRL.(ABSTRACT TRUNCATED AT 400 WORDS)

The effects of the polyamines putrescine (PUT), spermidine (SPD), and spermidine (SPM) on the secretion of plasminogen activator (PA) and plasminogen activator inhibitor (PAI) were evaluated using cultured bovine aortic endothelial cells. All three polyamines enhanced PA secretion in a time- and dose-dependent manner, with a potency rank order of SPM greater than SPD greater than PUT. The PA stimulation required both RNA and protein synthesis, as evidenced by inhibition of polyamine-induced PA secretion by actinomycin D and cycloheximide. The inhibitors of polyamine biosynthesis methylglyoxal bis-(guanylhydrazone) (MGBG) and dl-(difluoromethyl) ornithine (DFMO) alone did not affect basal or polyamine-induced PA secretion, with the exception that MGBG reduced the effect of PUT. Polyamine-treated cells enhanced secretions of both tissue-type and urokinase-type PA. The results of the present study suggest that polyamines may play a role in the regulation of PA synthesis and secretion and that this function can be modified under pathophysiological conditions affecting cellular and tissue levels of polyamines.

Decidual prolactin-releasing factor (PRL-RF), a placental protein that stimulates the release of prolactin from human decidual tissue, has been purified from conditioned medium of human placental explants. The purification scheme consisted of ethanol extraction, anion exchange chromatography on DEAE-cellulose, size exclusion chromatography on Spherogel TSK-3000, and either a) immunoaffinity chromatography using an antiserum to a partially purified PRL-RF preparation or b) acetic acid-urea/SDS 2-dimensional PAGE. The apparent molecular weight of the purified releasing factor, estimated by SDS-PAGE, was 23,500 Da; and the half-maximal dose for the acute stimulation of prolactin release from human decidual cells was 0.05-0.1 ug/ml (2.2-4.4 nM).

Previous studies from this laboratory provided evidence for the existence of a specific placental lactogen (PL) receptor in tissues of fetal lambs and pregnant sheep. The PL receptor is structurally and functionally distinct from somatotropic (GH) and lactogenic (PRL) receptors, and there are conspicuous differences in the expression of the three receptors during ontogeny. The results of the present study indicate striking differences in the solubilization of PL- and GH-binding sites in maternal and fetal sheep liver. Radiolabeled ovine PL (oPL) bound specifically and with high affinity (Kd, 0.97 nM) to soluble detergent extracts of ovine fetal liver, but there was no specific binding of radiolabeled ovine GH (oGH) or oPRL to soluble extracts or insoluble fractions of fetal liver. When liver microsomes of pregnant sheep were extracted with Triton X-100, 80% of the [125I]oPL-binding sites were recovered in the soluble fraction, but 76% of the [125I]oGH binding sites were recovered in the insoluble pellet. Soluble extracts of maternal liver had high affinity for oPL (Kd, 1.45 nM), but low affinity for oGH (Kd 33 nM) and oPRL (Kd, 1-2 microM). On the other hand, Triton-insoluble fractions of maternal liver had high affinity for oGH (Kd, 0.95 nM) as well as oPL (Kd, 0.91 nM), but low affinity for oPRL (Kd, 1-2 microM). The subunit structure of the [125I]oPL-binding site in soluble fractions of fetal and maternal liver (mol wt, 38-47K) was distinct from that of the [125I]oGH-binding site in Triton-insoluble fractions of maternal liver (mol wt, 54/118K). These findings indicate that treatment of microsomal fractions of fetal and maternal sheep liver with Triton X-100 solubilizes the oPL receptor but not the oGH receptor. The differential solubilization of PL- and GH-binding sites may facilitate purification of the two distinct receptors and clarification of their respective roles in the regulation of fetal and postnatal growth.