Assessment of bone dysplasia by micro-CT and glycosaminoglycan levels in mouse models for mucopolysaccharidosis type I, IIIA, IVA, and VII

Mucopolysaccharidoses (MPS) are a group of lysosomal storage diseases caused by mutations in lysosomal enzymes involved in degradation of glycosaminoglycans (GAGs). Patients with MPS grow poorly and become physically disabled due to systemic bone disease. While many of the major skeletal effects in mouse models for MPS have been described, no detailed analysis that compares GAGs levels and characteristics of bone by micro-CT has been done. The aims of this study were to assess severity of bone dysplasia among four MPS mouse models (MPS I, IIIA, IVA and VII), to determine the relationship between severity of bone dysplasia and serum keratan sulfate (KS) and heparan sulfate (HS) levels in those models, and to explore the mechanism of KS elevation in MPS I, IIIA, and VII mouse models. Clinically, MPS VII mice had the most severe bone pathology; however, MPS I and IVA mice also showed skeletal pathology. MPS I and VII mice showed severe bone dysplasia, higher bone mineral density, narrowed spinal canal, and shorter sclerotic bones by micro-CT and radiographs. Serum KS and HS levels were elevated in MPS I, IIIA, and VII mice. Severity of skeletal disease displayed by micro-CT, radiographs and histopathology correlated with the level of KS elevation. We showed that elevated HS levels in MPS mouse models could inhibit N-acetylgalactosamine-6-sulfate sulfatase enzyme. These studies suggest that KS could be released from chondrocytes affected by accumulation of other GAGs and that KS could be useful as a biomarker for severity of bone dysplasia in MPS disorders.     

Different response of murine and human mammary tumour models to a series of diastereoisomeric [1,2-bis(difluorophenyl) ethylenediamine]dichloroplatinum(II) complexes

Summary A series of isomeric [1,2-bis(difluorophenyl) ethylenediamine]dichloroplatinum(II) complexes and cisplatin were tested on the P388 leukemia and on the murine hormone-independent MXT (M3.2) OVEX and the ovarian — hormone — dependent MXT (M3.2) mammary carcinoma for evaluating antineoplastic activity against breast cancer in vivo. Although these results were heterogeneous, a trend to the 2,6-difiuorosubstituted compound as the most active platinum complex was observed. For the development of a large-scale in vitro screening method on human breast cancer cell lines, cell number, [³H]thymidine incorporation, and crystal violet staining were evaluated as parameters for end-point determination. Chemosensitivity testing on the human breast cancer cell lines MDA-MB-231 and MCF-7 unam-biguously identified [1,2-bis(2,4-difluorophenyl)ethyle-nediamine]dichloroplatinum(II) as the complex with the highest activity in the crystal violet micro-assay. In equimolar concentration this compound was superior to cisplatin on both cell lines. The analysis of the conflicting results of this study indicates that murine mammary carcinomas are most probably unrealistic and inappropriate models for the screening of cytotoxic platinum complexes with potential activity on human breast cancer.Abbreviations PBS phosphate-buffered saline - PEG polyethlyeneglycol Dedicated to Professor Franz Lux on the occasion of his 65th birthday     

Structure of hemoglobins Zürich [His E7(63)beta replaced by Arg] and Sydney [Val E11(67)beta replaced by Ala] and role of the distal residues in ligand binding.

In hemoglobin Zürich the side chain of the distal arginine attaches itself to the propionate of the heme, leaving the heme pocket wide open, allowing sulfanilamides easy access to the iron, and doubling the partition coefficient between CO and O2. The replacement of the distal valine by alanine in hemoglobin Sydney leaves a large gap inside the heme pocket, which is partly filled by a water molecule bonded to the distal histidine. Hemoglobin Sydney has the same partition coefficient between CO and O2 as hemoglobin A.Replacement of the distal histidine increases the stretching frequency of CO linked to the beta heme by 6 cm-1, but replacement of the distal valine increases it by only 3 cm-1, but replacement of the distal histidine leaves the O-O stretching frequency unchanged.     

Two new hemoglobin variants: Hb Brem-sur-Mer [beta9(A6)Ser-->Tyr] and Hb Passy [alpha81(F2)Ser-->Pro (alpha2)

Two new hemoglobin (Hb) variants: Hb Brem-sur-Mer [codon 9 (TCT-->TAT); beta9(A6)Ser-->Tyr] on the first exon of the beta-globin gene and Hb Passy [codon 81 (TCC-->CCC); alpha81(F2)Ser-->Pro (alpha2)] on the second exon of the alpha2-globin gene, are described. The two variants were characterized by DNA sequencing and mass spectrometry (MS). Hematological abnormalities: microcytosis and hypochromia were found only in the carrier of Hb Passy. In the absence of an association with an alpha-thalassemic deletion or mutation, the mutation 81(F2)Pro could induce a possible alpha-thalassemia (thal).     

Inhibition of angiogenesis and tumor growth by murine 7E3, the parent antibodyof c7E3 Fab (abciximab; ReoProTM)

Angiogenesis plays an essential role in the growth and dissemination of solid tumor cancers. The expression of endothelial cell integrin αvβ3 has been shown to increase during vascular proliferation associated with human tumors. Selective antagonists of αvβ3 can block angiogenesis and tumor growth by inducing programmed cell death in proliferating endothelial cells. Monoclonal antibody 7E3, an antagonist of the human, but not murine, integrins αvβ3 and αIIbβ3 (GPIIb/IIIa), inhibits platelet aggregation. It is the parent antibody of a mouse/human chimeric antibody fragment approved for adjunctive therapy of patients undergoing percutaneous coronary interventions to prevent ischemic complications (c7E3Fab; abciximab; ReoPro). To evaluate the potential of 7E3 to inhibit human angiogenesis and tumor growth independent of its antiplatelet effects, we established integrin αvβ3-negative human melanoma tumors in full-thickness human skin grafted onto SCID mice. The resulting tumors induce a human angiogenic response as assessed by the immunoreactivity of vascular cells with monoclonal antibodies specific for human CD31. Administration of 7E3 prevented or significantly inhibited the growth of tumors, and this effect correlated with a significant reduction in the number of blood vessels supplying the tumors. These results support the previous findings that blockade of integrin αvβ3 inhibits angiogenesis and tumor growth and indicates that dual inhibitors of αvβ3 and αIIbβ3 are effective in blocking tumor growth and angiogenesis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Beta-thalassemia intermedia in two Turkish families is caused by the interaction of Hb Knossos [beta 27(B9)Ala----Ser] and of Hb City of Hope [beta 69(E13)Gly----ser] with beta (0)-thalassemia

We have studied a few members of two Turkish families, who had a beta-thalassemia of the intermediate type. An abnormal hemoglobin was found in both families, which when present in association with beta(0)-thalassemia was considered to be the primary cause for the increased severity of the disease. In the first family this variant was Hb Knossos [beta 27(B9)Ala----Ser] which occurred together with the frameshift in codon #8 type of beta(0)-thalassemia. This compound heterozygosity, observed for the first time in the Turkish population was characterized by a considerable increase in Hb F production, mainly of the G gamma type, as expected for a chromosome with haplotype IV. In the second family, the variant was Hb City of Hope [beta 69(E13)Gly----Ser] which was present in combination with an unknown type of beta-thalassemia. The increase in Hb F production in the compound heterozygote was minimal. Reversed phase high performance liquid chromatography and the DNA amplification-synthetic oligonucleotide probe procedure were major tools in identifying the different abnormalities.     

Adenosine type 1 (A1) receptors mediate protection against myocardial infarction produced by chronic,intermittent ingestion of ethanol in dogs

BACKGROUND: Chronic consumption of small amounts of ethanol protects myocardium from ischemic injury. We tested the hypothesis that adenosine type 1 (A(1)) receptors mediate these beneficial effects. METHODS: Dogs (n=37) were fed with ethanol (1.5 g/kg) or water mixed with dry food twice per day for 12 weeks, fasted overnight before experimentation, and instrumented for measurement of hemodynamics. Dogs received intravenous drug vehicle (50% polyethylene glycol in 0.1 N sodium hydroxide and 0.9% saline over 15 min) or the selective A(1) receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.8 mg/kg over 15 min) and were subjected to a 60 min coronary artery occlusion followed by 3 h of reperfusion. Myocardial infarct size and transmural coronary collateral blood flow were measured with triphenyltetrazolium chloride staining and radioactive microspheres, respectively. RESULTS: The area at risk (AAR) for infarction was similar between groups. Pretreatment with ethanol significantly reduced infarct size to 13+/-2% (n=7) of the AAR as compared to control experiments (26+/-2%; n=7). DPCPX abolished the protective effects of ethanol pretreatment (30+/-3%; n=7) but had no effect in dogs that did not receive ethanol (25+/-2%; n=7). No differences in transmural coronary collateral blood flow were observed between groups. CONCLUSIONS: The present findings indicate that chronic ingestion of small amounts of ethanol produces myocardial protection that persists after the discontinuation of ethanol. The results indicate that A(1) receptors mediate ethanol-induced preconditioning in dogs independent of alterations in systemic hemodynamics or coronary collateral blood flow.     

Metabolic correction and cross-correction of mucopolysaccharidosis type II (Hunter syndrome) by retroviral-mediated gene transfer and expression of human iduronate-2-sulfatase.

To explore the possibility of using gene transfer to provide iduronate-2-sulfatase (IDS; EC 3.1.6.13) enzyme activity for treatment of Hunter syndrome, an amphotropic retroviral vector, L2SN, containing the human IDS coding sequence was constructed and studied for gene expression in vitro. Lymphoblastoid cell lines (LCLs) from patients with Hunter syndrome were transduced with L2SN and expressed high levels of IDS enzyme activity, 10- to 70-fold higher than normal human peripheral blood leukocytes or LCLs. Such L2SN-transduced LCLs failed to show accumulation of 35SO4 into glycosaminoglycan (35SO4-GAG), indicating that recombinant IDS enzyme participated in GAG metabolism. Coculture of L2SN-transduced LCLs with fibroblasts from patients with Hunter syndrome reduced the accumulation of 35SO4-GAG. These results demonstrated retroviral-mediated IDS gene transfer into lymphoid cells and the ability of such cells to provide recombinant enzyme for intercellular metabolic cross-correction.     

Tsg101, a homologue of ubiquitin-conjugating (E2) enzymes, binds the L domain in HIV type 1 Pr55Gag

Ubiquitination appears to be involved in virus particle release from infected cells. Free ubiquitin (Ub), as well as Ub covalently bound to a small fraction of p6 Gag, is detected in mature HIV particles. Here we report that the p6 region in the Pr55(Gag) structural precursor polyprotein binds to Tsg101, a putative Ub regulator that is involved in trafficking of plasma membrane-associated proteins. Tsg101 was found to interact with Gag in (i) a yeast two-hybrid assay, (ii) in vitro coimmunoprecipitation by using purified Pr55(Gag) and rabbit reticulocyte lysate-synthesized Tsg101, and (iii) in vivo in the cytoplasm of COS cells transfected with gag. The PTAPP motif [or late (L) domain] within p6, which is required for release of mature virus from the plasma membrane, was the determinant for binding Pr55(Gag). The N-terminal region in Tsg101, which is homologous to the Ubc4 class of Ub-conjugating (E2) enzymes, was the determinant of interaction with p6. Mutation of Tyr-110 in Tsg101, present in place of the active-site Cys that binds Ub in E2 enzymes, and other residues unique to Tsg101, impaired p6 interaction, indicating that features that distinguish Tsg101 from active E2 enzymes were important for binding the viral protein. The results link L-domain function in HIV to the Ub machinery and a specific component of the cellular trafficking apparatus.     

Localization of medullary thyroid carcinoma metastasis in a multiple endocrine neoplasia type 2A patient by 6-[18F]-fluorodopamine positron emission tomography

6-[(18)F]fluorodopamine, a substrate for the norepinephrine transporter, has been used as a tumor-seeking tracer in positron emission tomography (PET) to localize pheochromocytomas and other chromaffin tumors. Here, we report the case of a 42-yr-old woman with multiple endocrine neoplasia type 2A, in whom biopsy-proven recurrent medullary thyroid cancer (MTC) was detected by 6-[(18)F]fluorodopamine PET scanning. The patient had previously undergone bilateral adrenalectomy for pheochromocytoma, total thyroidectomy, and extirpation of a parapharyngeal MTC metastatic deposit. An increase in plasma calcitonin 5 yr after her initial presentation was further investigated, leading to the discovery of a mass in the left parapharyngeal space. Levels of serum and urine catecholamines and metanephrines were normal. To exclude a hormonally silent pheochromocytoma metastasis, 6-[(18)F]fluorodopamine PET was performed. The study showed a focus of radionuclide accumulation corresponding to the parapharyngeal mass. After resection of the latter, pathology confirmed metastatic MTC. To our knowledge, this is the first case of metastatic, histologically proven MTC, which was unequivocally detected by 6-[(18)F]fluorodopamine PET scanning. Because norepinephrine transporter systems have been previously found in MTC, it is conceivable that 6-[(18)F]fluorodopamine PET scanning can be used for the diagnostic localization of this tumor and its metastatic deposits because total and early resection is beneficial to the outcome of the patient.     

Molecular characterization of Hb D-Ibadan [beta87(F3)Thr-->Lys] in combination with Hb S [beta6(A3)Glu-->Val] and with beta+-Thalassemia: report of two cases

Hb D-Ibadan [beta87(F3)Thr-->Lys] is a common variant in the Nigerian population, which has been reported in association with Hb S [beta6(A3)Glu-->Val] and with beta-thalassemia. Unlike the Hb S/Hb D-Los Angeles [beta121(GH4)Glu-->Gln] combination, compound heterozygosity for Hb D-Ibadan and Hb S does not result in a sickling disorder. We report the first case of a combination of Hb D-Ibadan with beta+-thalassemia, and the first observation of Hb S/Hb D-Ibadan in the African-American population. In both cases, the characterization of Hb D-Ibadan was achieved by sequencing of the genomic DNA. Although protein based methods such as isoelec-trofocusing and high performance liquid chromatography may suggest that the "D-like" variant is different from Hb D-Los Angeles, the definitive identification of the variant by structural analysis or molecular genetic methods should be undertaken, particularly in newborn screening programs when the variant is found in combination with Hb S.     

Complex interaction of Hb Hekinan [alpha27(B8) Glu-Asp] and Hb E [beta26(B8) Glu-Lys] with a deletional alpha-thalassemia 1 in a Thai family

Hemoglobin (Hb) Hekinan (alpha27; Glu-Asp) is a rare alpha-chain variant found mainly in Japanese and Chinese whereas Hb E (beta26; Glu-Lys) is common among Southeast Asians. We report a hitherto undescribed condition in which these two variants co-segregate. The proband was a 25-yr-old Thai woman who was encountered with the presence of mild hypochromic microcytosis with Hb 8.2 g/dL, hematocrit (Hct) 26.0%, Mean Corpuscular Value (MCV) 68.6 fL, Mean Corpuscular Hemoglobin (MCH) 21.6 pg and Mean Corpuscular Hemoglobin Concentration (MCHC) 31.5 g/dL. Although Hb electrophoresis at alkaline pH did not show any abnormal band except Hb E in addition to Hb A, high performance liquid chromatography analysis revealed abnormal peaks at the Hb A and Hb E positions. DNA analysis of the proband revealed a GAG-GAT mutation at codon 27 of the minor alpha1-globin gene for Hb Hekinan in trans to the South-east Asian (SEA) deletional alpha-thalassemia 1 determinant and a GAG-AAG mutation at codon 26 of the beta-globin gene for Hb E. She was therefore a triple heterozygote for these three anomalies. Family study identified that her mother was a double heterozygote for Hb Hekinan and Hb E without alpha-thalassemia whereas her father was a classical Hb H disease patient. The genotype-phenotype relationship observed in this Thai family with complex hemoglobinopathies is presented and a simple DNA assay based on the polymerase chain reaction methodology for rapid diagnosis of Hb Hekinan is described.     

Scavenging effect of lazaroids U78517F,U74389F,U74500A on oxygen free radicals produced by stimulated human polymorphonuclear leucocytes and by chemical method

Authors investigated the scavenger capability of lazaroids, a new group of compounds (21-aminosteroid) that are reported in the literature to have interesting anti-lipid peroxidation properties. Authors tested the degree of scavenger activity related to the oxygen derived free radicals (ODFR) with different methods: 1) chemiluminescence; 2) production of superoxide anion from activated polymorphonuclear cells; 3) production of hydroxyl radical through a chemical procedure. Results showed a global scavenger activity of the three lazaroids (U78517F, U74389F, U74500A) in all the various tests, but differences of intensity of their action were noted among in each compound. We can thus attribute to these compounds a scavenger activity on the oxygen free radicals; this activity may facilitate their already known anti-lipid peroxidation action. Therefore, clinical use of lazaroids can be hypothesized for the diseases in which inflammation plays an important pathogenetic role via the production of oxygen free radicals and the resulting lipid peroxidation associated with tissue damage.