Phytanic acid α-oxidase deficiency (Refsum disease) presenting in infancy

Summary This report describes a patient with high serum phytanic acid concentration due to phytanic acid -oxidase deficiency (classical Refsum disease). He presented unusually early, hypotonia and developmental delay being apparent by 7 months. A generalized peroxisomal disorder (so-called infantile Refsum disease) was excluded by analyses of pristanic acid, very long-chain fatty acids, bile acids and plasmalogen synthesis. The early presentation raises the possibility ofin utero exposure to phytanate.     

A novel α-thalassemia frameshift mutation: codon 8 (-C)

We report a novel α-thalassemia (α-thal) point mutation detected during newborn screening for hemoglobinopathies. Sequence analyses identified a frameshift mutation at codon 8 (-C) in exon 1 of the α2-globin gene. This mutation causes an α(+)-thal phenotype.     

Heteromerization of chemokine (C-X-C motif) receptor 4 with α1A/B-adrenergic receptors controls α1-adrenergic receptor function

Recent evidence suggests that chemokine (C-X-C motif) receptor 4 (CXCR4) contributes to the regulation of blood pressure through interactions with α₁-adrenergic receptors (ARs) in vascular smooth muscle. The underlying molecular mechanisms, however, are unknown. Using proximity ligation assays to visualize single-molecule interactions, we detected that α_1A/B-ARs associate with CXCR4 on the cell surface of rat and human vascular smooth muscle cells (VSMC). Furthermore, α_1A/B-AR could be coimmunoprecipitated with CXCR4 in a HeLa expression system and in human VSMC. A peptide derived from the second transmembrane helix of CXCR4 induced chemical shift changes in the NMR spectrum of CXCR4 in membranes, disturbed the association between α_1A/B-AR and CXCR4, and inhibited Ca²⁺ mobilization, myosin light chain (MLC) 2 phosphorylation, and contraction of VSMC upon α₁-AR activation. CXCR4 silencing reduced α_1A/B-AR:CXCR4 heteromeric complexes in VSMC and abolished phenylephrine-induced Ca²⁺ fluxes and MLC2 phosphorylation. Treatment of rats with CXCR4 agonists (CXCL12, ubiquitin) reduced the EC₅₀ of the phenylephrine-induced blood pressure response three- to fourfold. These observations suggest that disruption of the quaternary structure of α_1A/B-AR:CXCR4 heteromeric complexes by targeting transmembrane helix 2 of CXCR4 and depletion of the heteromeric receptor complexes by CXCR4 knockdown inhibit α₁-AR–mediated function in VSMC and that activation of CXCR4 enhances the potency of α₁-AR agonists. Our findings extend the current understanding of the molecular mechanisms regulating α₁-AR and provide an example of the importance of G protein-coupled receptor (GPCR) heteromerization for GPCR function. Compounds targeting the α_1A/B-AR:CXCR4 interaction could provide an alternative pharmacological approach to modulate blood pressure.Chemokine (C-X-C motif) receptor 4 (CXCR4) is a G protein-coupled receptor (GPCR) that is essential during development. Animals lacking CXCR4 are not viable and demonstrate defects of the hematopoietic and cardiovascular system (1). After birth, CXCR4 is expressed in many tissues, including the heart and vasculature, and fulfills multiple functions in the immune system, such as regulation of leukocyte trafficking, stem cell mobilization, and homing (2, 3). Moreover, CXCR4 is involved in various disease processes, such as HIV infection, cancer metastasis, and tissue repair (3–5).In addition to these established functions, recent observations suggest that CXCR4 also contributes to the regulation of hemodynamics and blood pressure. Treatment with the CXCR4 antagonists AMD3100 and AMD3465 reduced blood pressure in experimental models of pulmonary arterial and systemic hypertension (6, 7). We have shown previously that AMD3100 reduces hemodynamic stability and blood pressure during the cardiovascular stress response to traumatic and hemorrhagic shock, whereas selective activation of CXCR4 with the noncognate agonist ubiquitin improves hemodynamic stability and increases systemic blood pressure after traumatic, hemorrhagic, and endotoxic shock (8–13). Because in vivo pharmacological targeting of CXCR4 did not affect myocardial function, these findings suggested that effects of CXCR4 on hemodynamics and blood pressure are mediated via modulation of vascular function (9). Accordingly, we observed that CXCR4 activation enhances and sensitizes vasoconstriction of isolated mesenteric arteries and veins in response to α₁-adrenergic receptor (AR) activation with phenylephrine (PE) (9). As these effects were independent of the vascular endothelium, interactions between CXCR4 and α₁-AR in vascular smooth muscle likely constitute the physiological basis for these observations (9). The molecular mechanisms underlying interactions between CXCR4 and α₁-AR in vascular smooth muscle, however, remain unknown.Crosstalk between GPCRs is a widely recognized principle that expands the physiological repertoire of GPCR-mediated signaling events and functions (14–19). Receptor crosstalk can be attributed to a variety of molecular mechanisms, including receptor hetero-oligomerization (14–23). The formation of homo- and/or hetero-oligomeric complexes among GPCRs is thought to be important for many aspects of GPCR function (22–24).CXCR4 has been shown to associate with multiple chemokine receptors in various expression systems (3, 25–28). ARs are also known to be able to form heteromeric receptor complexes (29–35), and recent evidence suggests that AR may also be able to form heteromeric complexes with chemokine receptors (36–38). Thus, we studied whether α₁-AR and CXCR4 may interact on the cell surface of vascular smooth muscle cells through the formation of heteromeric receptor complexes.Here, we provide evidence that heteromeric receptor complexes between α_1A-AR and CXCR4 and between α_1B-AR and CXCR4 are constitutively expressed in rat and human vascular smooth muscle cells (VSMC). We show that disruption of the quaternary structure of the heteromeric receptor complex by targeting transmembrane helix (TM) 2 of CXCR4 and depletion of heteromeric receptor complexes by CXCR4 knockdown inhibit α₁-AR agonist-induced key signaling events and contraction of VSMC. Furthermore, we show that treatment with CXCR4 agonists increases the potency of the α₁-AR agonist PE to increase blood pressure in vivo. Our observations suggest that α₁-AR function in VSMC is controlled through the formation of heteromeric α_1A/B-AR:CXCR4 complexes.     

A Novel α-Thalassemia Frameshift Mutation: Codon 8 (–C)

We report a novel α-thalassemia (α-thal) point mutation detected during newborn screening for hemoglobinopathies. Sequence analyses identified a frameshift mutation at codon 8 (–C) in exon 1 of the α2-globin gene. This mutation causes an α⁺-thal phenotype.     

Hb Moriguchi or α2β297(FG4)His→Tyr Substitution at the α1-−β2 Interface

An abnormal profile of cation exchange high performance liquid chromatography for the determination of Hb A_1c led to the discovery of a new hemoglobin variant with a His→Tyr substitution at position 97(FG4) of the β chain. The variant comprised about equal proportion to normal Hb A. Lt showed an increased oxygen affinity, normal n value and slightly reduced alkaline Bohr effect. However, the magnitude of the functional deviation is less than that of other variants with substitutions at the same position, and the heterozygous carrier was not erythremic.     

Construction, expression and characterization of human interferon α2b-(G4S)n-thymosin α1 fusion proteins in Pichia pastoris

AIM: Interferon α2b (IFNα2b) and thymosin α1 (Tα1) exhibit synergic effects in the treatment of hepatitis B and hepatitis C when used together. For developing a fusion protein drug, fusion proteins of IFNα2b and Ta1 linked by different lengths of (G4S)n(n = 1-3) were constructed and expressed in Pichia pastoris. METHODS: Using PCR and molecular clone techniques, the fusion genes of IFNα2b-(G4S)n-Tα1 (n = 1-3) were constructed and subcloned into the eukaryotic expression vector pPIC9. After transformation of these plasmids into P. pastoris, the expressed fusion proteins IFNα2b-(G4S) n-Tα1 (n = 1-3) were obtained. These proteins were purified through diethylaminoethyl (DEAE) affinity chromatography and Superdex?75 gel filtration and analyzed by SDS-PAGE and Western blot. Antiviral and E-rosette assays were used to investigate the bioactivities of these fusion proteins. RESULTS: DNA sequencing confirmed that the fusion genes of IFNa2b-(G4S)n-Tα1 (n= 1-3) were correctly cloned to the pPIC9 vector. The recombinant IFNα2b-(G4S)n-Tα1 (n = 1-3) fusion proteins expressed in P. pastoris were purified with DEAE and Superdex?75 gel filtration chromatography. The fusion proteins could be observed on sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular weight (MW) of 23.2, 22.9, and 22.6 ku, respectively, and reacted to the IFNa2b monoclonal antibody and Tal polyclonal antibody. The purified fusion proteins exhibit antiviral activity and can enhance the percentage of E-rosette-forming-cell in E-rosette assay. CONCLUSION: The recombinant IFNa2b-(G4S)n-Tα1 (n = 1-3) fusion proteins were successfully expressed in P. pastoris. Purified fusion proteins exhibit both antiviral activity of IFNa2b and immunomodulatory activity of Tal in vitro. These results will be the basis for further evaluation of the fusion proteins' function in vivo.     

A potential role for α-actinin in inside-out αIIbβ3 signaling

Many different biochemical signaling pathways regulate integrin activation through the integrin cytoplasmic tail. Here, we describe a new role for α-actinin in inside-out integrin activation. In resting human platelets, α-actinin was associated with αIIbβ3, whereas inside-out signaling (αIIbβ3 activation signals) from protease-activated receptors (PARs) dephosphorylated and dissociated α-actinin from αIIbβ3. We evaluated the time-dependent changes of the αIIbβ3 activation state by measuring PAC-1 binding velocity. The initial velocity analysis clearly showed that PAR1-activating peptide stimulation induced only transient αIIbβ3 activation, whereas PAR4-activating peptide induced long-lasting αIIbβ3 activation. When αIIbβ3 activation signaling dwindled, α-actinin became rephosphorylated and reassociated with αIIbβ3. Compared with control platelets, the dissociation of α-actinin from αIIbβ3 was only transient in PAR4-stimulated P2Y(12)-deficient platelets in which the sustained αIIbβ3 activation was markedly impaired. Overexpression of wild-type α-actinin, but not the mutant Y12F α-actinin, increased its binding to αIIbβ3 and inhibited PAR1-induced initial αIIbβ3 activation in the human megakaryoblastic cell line, CMK. In contrast, knockdown of α-actinin augmented PAR-induced αIIbβ3 activation in CMK. These observations suggest that α-actinin might play a potential role in setting integrins to a default low-affinity ligand-binding state in resting platelets and regulating αIIbβ3 activation by inside-out signaling.     

4 α1-Antitrypsin deficiency

α₁-Antitrypsin deficiency (PiZ) is frequent in Caucasian populations. The predominant clinical correlates of this inborn error, i.e. chronic liver disease, emphysema, and vasculitic syndromes including their pathogenetic background are discussed in the present review.     

Hb Lynwood [α107(G14) (-T) (α2) HBA2:c.323delT)] in conjunction with the α(3.7) deletion produces a moderately severe α-thalassemia phenotype

We describe a novel frameshift mutation associated with an α-thalassemia (α-thal) phenotype in a patient of Sudanese origin investigated for persistent microcytosis. In addition to the α(3.7) deletion, a novel mutation on the α2 gene was detected: HBA2:c.323delT. This mutation causes a frameshift at codon 107 of the α2 gene. The result is a disturbed amino acid sequence for the following 24 amino acids, and a premature termination codon at position 132.     

Effects of human α-interferon on granulocyte-macrophage progenitor cells (CFU-GM) in vitro

Summary Two preparations of human interferon (IFN)- were assessed for their influence on granulocyte-macrophage progenitor cells (CFU-GM) in vitro. Both highly purified human IFN- Ly and recombinant IFN- 2a suppressed CFU-GM colony formation in a dose-dependent manner using low-density bone-marrow target cells. Suppression of CFU-GM colony formation was accompanied by an increase in clusters. However, depletion of monocytes, T lymphocytes and B lymphocytes from low-density bone-marrow cells resulted in insensitivity of progenitor cells to IFN-. These results demonstrate that the effects of human IFN- on myeloid progenitor cells (CFU-GM) are mediated by accessory cells within the bone marrow.     

INTERACTION OF THE α2 POLYADENYLATION SIGNAL MUTATION (AATAAA → AATA– –) AND α0-THALASSEMIA (– –SEA), RESULTING IN Hb H DISEASE IN A THAI PATIENT

We report a Thai boy with a compound heterozygosity for the α2 polyadenylation signal mutation (AATAAA → AATA– –) and α⁰-thalassemia (– –^SEA), who suffered from Hb H disease with more severe clinical symptoms than those usually observed with deletional Hb H disease. His Hb H level was as high as 52% of total hemoglobin. The hematologic data of this unusual case of Hb H disease was compared with those of Hb H disease with a homozygosity for the α2 polyadenylation signal mutation, and compound heterozygosity of the α2 polyadenylation signal mutation and α⁰-thalassemia. A simple DNA assay based on an allele specific polymerase chain reaction for the detection of this polyadenylation signal mutation is described.     

Two novel mutations in the gene for human α-mannosidase that cause α-mannosidosis

Mutation analysis performed on two Italian patients with alpha-mannosidosis allowed the identification of two new mutations, IVS20-2A>G and 322-323insA. The patients were both homozygous for these mutations. The first mutation causes skipping of exon 21, whereas the second causes a frameshift introducing a stop codon at position 160 of the amino acid sequence.     

Hb J-Wenchang-Wuming [α11(A9)Lys→Gln (AAG>CAG) (α2 or α1)] compromises neonatal screening for α-thalassemia with the Sebia Capillarys2 electrophoresis system

We describe a Chinese newborn who was assumed to have α(0)-thalassemia (α(0)-thal) by determining the amount of Hb Bart's (γ4) in the cord blood, but was later shown to have only α(+)-thal. Hb J-Wenchang-Wuming [α11(A9)Lys→Gln (AAG>CAG) (α2 or α1)] was mistaken for Hb Bart's as both hemoglobin (Hb) variants have the same mobility.