Correction of murine galactosialidosis by bone marrow-derived macrophages overexpressing human protective protein/cathepsin A under control of the colony-stimulating factor-1 receptor promoter

Galactosialidosis (GS) is a human neurodegenerative disease caused by a deficiency of lysosomal protective protein/cathepsin A (PPCA). The GS mouse model resembles the severe human condition, resulting in nephropathy, ataxia, and premature death. To rescue the disease phenotype, GS mice were transplanted with bone marrow from transgenic mice overexpressing human PPCA specifically in monocytes/macrophages under the control of the colony stimulating factor-1 receptor promoter. Transgenic macrophages infiltrated and resided in all organs and expressed PPCA at high levels. Correction occurred in hematopoietic tissues and nonhematopoietic organs, including the central nervous system. PPCA-expressing perivascular and leptomeningeal macrophages were detected throughout the brain of recipient mice, although some neuronal cells, such as Purkinje cells, continued to show storage and died. GS mice crossed into the transgenic background reflected the outcome of bone marrow-transplanted mice, but the course of neuronal degeneration was delayed in this model. These studies present definite evidence that macrophages alone can provide a source of corrective enzyme for visceral organs and may be beneficial for neuronal correction if expression levels are sufficient.

Correction of hypertension by normalization of endothelial levels of fibroblast growth factor and nitric oxide synthase in spontaneously hypertensive rats.

Acidic and basic fibroblast growth factors (FGFs) share a wide range of diverse biological activities. To date, low levels of FGF have not been correlated with a pathophysiologic state. We report that blood vessels of spontaneously hypertensive rats are shown to be associated with a marked decrement in endothelial basic FGF content. This decrement correlates both with hypertension and with a decrease in the endothelial content of nitric oxide synthase. Restoration of FGF to physiological levels in the vascular wall, either by systemic administration or by in vivo gene transfer, significantly augmented the number of endothelial cells with positive immunostaining for nitric oxide synthase, corrected hypertension, and ameliorated endothelial-dependent responses to vasoconstrictors. These results suggest an important role for FGFs in blood pressure homeostasis and open new avenues for the understanding of the etiology and treatment of hypertension.

SoxR, a [2Fe-2S] transcription factor, is active only in its oxidized form.

SoxR protein is known to function both as a sensor and as a transcriptional activator for a superoxide response regulon in Escherichia coli. The activity of SoxR was tested by its ability to enable the transcription of its target gene, soxS, in vitro. The activity of the oxidized form was lost when its [2Fe-2S] clusters were reduced by dithionite under anaerobic conditions, and it was rapidly restored by autooxidation. This result is consistent with the hypothesis that induction of the regulon is effected by the univalent oxidation of the Fe-S centers of SoxR. In vivo, this oxidation may be caused by an alteration of the redox balance of electron chain intermediates that normally maintains soxR in an inactive, reduced state. Oxidized SoxR was about twice as effective as reduced SoxR in protecting the soxS operator from endonucleolytic cleavage. However, this difference could not account for a greater than 50-fold difference in their activities and therefore could not support a model in which oxidation activates SoxR by enabling it to bind to DNA. NADPH, ferredoxin, flavodoxin, or ferredoxin (flavodoxin):NADP+ reductase could not reduce SoxR directly in vitro at a measurable rate. The midpoint potential for SoxR was measured at -283 mV.

Thymus transplantation, a critical factor for correction of autoimmune disease in aging MRL/+mice.

MRL/MP-+/+ (MRL/+) mice develop pancreatitis and sialoadenitis after they reach 7 months of age. Conventional bone marrow transplantation has been found to be ineffective in the treatment of these forms of apparent autoimmune disease. Old MRL/+ mice show a dramatic thymic involution with age. Hematolymphoid reconstitution is incomplete when fetal liver cells (as a source of hemopoietic stem cells) plus fetal bone (FB; which is used to recruit stromal cells) are transplanted from immunologically normal C57BL/6 donor mice to MRL/+ female recipients. Embryonic thymus from allogeneic C57BL/6 donors was therefore engrafted along with either bone marrow or fetal hematopoietic cells (FHCs) plus fragments of adult or fetal bone. More than seventy percent of old MRL/+ mice (> 7 months) that had been given a fetal thymus (FT) transplant plus either bone marrow or FHCs and also bone fragments survived more than 100 days after treatment. The mice that received FHCs, FB, plus FT from allogeneic donors developed normal T cell and B cell functions. Serum amylase levels decreased in these mice whereas they increased in the mice that received FHCs and FB but not FT. The pancreatitis and sialoadenitis already present at the time of transplantations were fully corrected according to histological analysis by transplants of allogeneic FHCs, FB and FT in the MRL/+ mice. These findings are taken as an experimental indication that perhaps stem cell transplants along with FT grafts might represent a useful strategy for treatment of autoimmune diseases in aged humans.

Matrix metalloproteinase 2 releases active soluble ectodomain of fibroblast growth factor receptor 1.

Recent studies have demonstrated the existence of a soluble fibroblast growth factor (FGF) receptor type 1 (FGFR1) extracellular domain in the circulation and in vascular basement membranes. However, the process of FGFR1 ectodomain release from the plasma membrane is not known. Here we report that the 72-kDa gelatinase A (matrix metalloproteinase type 2, MMP2) can hydrolyze the Val368-Met369 peptide bond of the FGFR1 ectodomain, eight amino acids upstream of the transmembrane domain, thus releasing the entire extracellular domain. Similar results were obtained regardless of whether FGF was first bound to the receptor or not. The action of MMP2 abolished binding of FGF to an immobilized recombinant FGFR1 ectodomain fusion protein and to Chinese hamster ovary cells overexpressing FGFR1 The released recombinant FGFR1 ectodomain was able to bind FGF after MMP2 cleavage, suggesting that the cleaved soluble receptor maintained its FGF binding capacity. The activity of MMP2 could not be reproduced by the 92-kDa gelatinase B (MMP9) and was inhibited by tissue inhibitor of metalloproteinase type 2. These studies demonstrate that FGFR1 may be a specific target for MMP2 on the cell surface, yielding a soluble FGF receptor that may modulate the mitogenic and angiogenic activities of FGF.

The recombinant proregion of transforming growth factor beta1 (latency-associated peptide) inhibits active transforming growth factor beta1 in transgenic mice.

All three isoforms of transforming growth factors beta (TGF-betal, TGF-beta2, and TGF-beta3) are secreted as latent complexes and activated extracellularly, leading to the release of the mature cytokines from their noncovalently associated proregions, also known as latency-associated peptides (LAPs). The LAP region of TGF-beta1 was expressed in a baculovirus expression system and purified to homogeneity. In vitro assays of growth inhibition and gene induction mediated by TGF-beta3 demonstrate that recombinant TGF-beta1 LAP is a potent inhibitor of the activities of TGF-betal, -beta2, and -beta3. Effective dosages of LAP for 50% neutralization of TGF-beta activities range from 4.7- to 80-fold molar excess depending on the TGF-beta isoform and activity examined. Using 125I-labeled LAP, we show that the intraperitoneal application route is effective for systemic administration of LAP. Comparison of concentrations of LAP in tissues shows a homogenous pattern in most organs with the exception of heart and muscle, in which levels of LAP are 4- to 8-fold lower. In transgenic mice with elevated hepatic levels of bioactive TGF-betal, treatment with recombinant LAP completely reverses suppression of the early proliferative response induced by TGF-beta1 in remnant livers after partial hepatectomy. The results suggest that recombinant LAP is a potent inhibitor of bioactive TGF-beta both in vitro and in vivo, after intraperitoneal administration. Recombinant LAP should be a useful tool for novel approaches to study and therapeutically modulate pathophysiological processes mediated by TGF-beta3.

Constitutively active human Notch1 binds to the transcription factor CBF1 and stimulates transcription through a promoter containing a CBF1-responsive element.

Notch is a transmembrane receptor that plays a critical role in cell fate determination. In Drosophila, Notch binds to and signals through Suppressor of Hairless. A mammalian homologue of Suppressor of Hairless, named CBF1 (or RBPJk), is a ubiquitous transcription factor whose function in mammalian Notch signaling is unknown. To determine whether mammalian Notch can stimulate transcription through a CBF1-responsive element (RE), we cotransfected a CBF1-RE-containing chloramphenicol acetyltransferase reporter and N1(deltaEC), a constitutively active form of human Notch1 lacking the extracellular domain, into DG75, COS-1, HeLa, and 293T cells, which all contain endogenous CBF1. N1(deltaEC) dramatically increased chloramphenicol acetyltransferase activity in these cells, indicating functional coupling of Notch1 and CBF1. The activity was comparable to that produced by the Epstein-Barr virus protein EBNA2, a well-characterized, potent transactivator of CBF1. To test whether CBF1 and Notch1 interact physically, we tagged CBF1 with an epitope from the influenza virus hemagglutinin or with the N-terminal domain of gal4, and transfected the tagged CBF1 plus N1(deltaEC) into COS-1 cells. Cell lysates were immunoprecipitated and immunoblotted with several anti-Notch1 antibodies [to detect N1(deltaEC)] or with antibodies to hemagglutinin or gal4 (to detect CBF1). Each immunoprecipitate contained a complex of N1(deltaEC) and CBF1. In summary, we find that the truncated, active form of human Notch1, N1(deltaEC), binds CBF1 and activates transcription through a CBF1-RE-containing promoter. We conclude that CBF1 is a critical downstream protein in the human Notch1 signaling pathway.

X-ray structure of clotting factor IXa: active site and module structure related to Xase activity and hemophilia B.

Hereditary deficiency of factor IXa (fIXa), a key enzyme in blood coagulation, causes hemophilia B, a severe X chromosome-linked bleeding disorder afflicting 1 in 30,000 males; clinical studies have identified nearly 500 deleterious variants. The x-ray structure of porcine fIXa described here shows the atomic origins of the disease, while the spatial distribution of mutation sites suggests a structural model for factor X activation by phospholipid-bound fIXa and cofactor VIIIa. The 3.0-A-resolution diffraction data clearly show the structures of the serine proteinase module and the two preceding epidermal growth factor (EGF)-like modules; the N-terminal Gla module is partially disordered. The catalytic module, with covalent inhibitor D-Phe-1I-Pro-2I-Arg-3I chloromethyl ketone, most closely resembles fXa but differs significantly at several positions. Particularly noteworthy is the strained conformation of Glu-388, a residue strictly conserved in known fIXa sequences but conserved as Gly among other trypsin-like serine proteinases. Flexibility apparent in electron density together with modeling studies suggests that this may cause incomplete active site formation, even after zymogen, and hence the low catalytic activity of fIXa. The principal axes of the oblong EGF-like domains define an angle of 110 degrees, stabilized by a strictly conserved and fIX-specific interdomain salt bridge. The disorder of the Gla module, whose hydrophobic helix is apparent in electron density, can be attributed to the absence of calcium in the crystals; we have modeled the Gla module in its calcium form by using prothrombin fragment 1. The arched module arrangement agrees with fluorescence energy transfer experiments. Most hemophilic mutation sites of surface fIX residues occur on the concave surface of the bent molecule and suggest a plausible model for the membrane-bound ternary fIXa-FVIIIa-fX complex structure: fIXa and an equivalently arranged fX arch across an underlying fVIIIa subdomain from opposite sides; the stabilizing fVIIIa interactions force the catalytic modules together, completing fIXa active site formation and catalytic enhancement.

New Approach for Correction of Error Associated With Keratometric Estimation of Corneal Power in Keratoconus

The aim of this study was to obtain the exact value of the keratometric index (nkexact) and to clinically validate a variable keratometric index (nkadj) that minimizes this error. Methods: The nkexact value was determined by obtaining differences (DPc) between keratometric corneal power (Pk) and Gaussian corneal power (PGauss c ) equal to 0. The nkexact was defined as the value associated with an equivalent difference in the magnitude of DPc for extreme values of posterior corneal radius (r2c) for each anterior corneal radius value (r1c). This nkadj was considered for the calculation of the adjusted corneal power (Pkadj). Values of r1c ∈ (4.2, 8.5) mm and r2c ∈ (3.1, 8.2) mm were considered. Differences of True Net Power with PGauss c , Pkadj, and Pk(1.3375) were calculated in a clinical sample of 44 eyes with keratoconus. Results: nkexact ranged from 1.3153 to 1.3396 and nkadj from 1.3190 to 1.3339 depending on the eye model analyzed. All the nkadj values adjusted perfectly to 8 linear algorithms. Differences between Pkadj and PGauss c did not exceed 60.7 D (Diopter). Clinically, nk = 1.3375 was not valid in any case. Pkadj and True Net Power and Pk(1.3375) and Pkadj were statistically different (P , 0.01), whereas no differences were found between PGauss c and Pkadj (P . 0.01). Conclusions: The use of a single value of nk for the calculation of the total corneal power in keratoconus has been shown to be imprecise, leading to inaccuracies in the detection and classification of this corneal condition. Furthermore, our study shows the relevance of corneal thickness in corneal power calculations in keratoconus.

The Economy factor in steam-power plants /

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