A new hemoglobin variant found during Hb A1c measurement: Hb Hokusetsu [beta52(D3)Asp-->Gly]

A new beta chain variant was accidentally found through the assay of Hb A1c in a diabetic patient. The variant was detected by polyacrylamide gel isoelectrofocusing and electrospray ionization mass spectrometry. For sequence determination, globin was cleaved with combination of trypsin and lysyl endopeptidase and analyzed by high performance liquid chromatography connected to electrospray ionization mass spectrometry. An abnormal betaT-5 peptide was found by reconstructed selected ion monitoring. The collision-induced dissociation spectrum of an ion derived from the abnormal betaT-5 peptide revealed a new substitution, [beta52(D3)Asp-->Gly], named Hb Hokusetsu. The sequence was confirmed with an automatic sequencer using peptides isolated by reversed phase high performance liquid chromatography. Amplification of the beta-globin exon 2 and nucleotide sequencing revealed a GAT-->GGT mutation in codon 52 corresponding to an Asp-->Gly replacement. Electrospray ionization mass spectrometry analysis of the hemolysate showed a reasonable value of 10.4% for glycated globin. The variant migrated as Hb S on isoelectrofocusing. Hematological analysis revealed normal parameters. The patient's hemolysate showed normal stability in the isopropanol test. Oxygen equilibrium studies on the patient's red blood cells and hemolysate showed no significant change in oxygen affinity or cooperativity.     

Hb Valletta [beta 87(F3)Thr-->Pro] due to an A-->C substitution at codon 87 in a Calabrian family with alpha-thalassemia

Genetic effects were studied in house mice caught from 1986 to 1994 in regions polluted by radionuclides as a result of the Chernobyl disaster. The dose rates of gamma-radiation on the soil surface ranged from 0.0002 to 2 mGy/h. The frequency of reciprocal translocations in mouse spermatocytes was relatively low, but increased with the dose rate. Embryo mortality was increased only in the progeny of male mice in males caught in 1987 in the area with maximal contamination. The frequency of mice heterozygous for recessive lethal mutations decreased with time after the accident.     

Hb Helsinki: a variant with a high oxygen affinity and a substitution at a 2,3-DPG binding site (beta82[EF6] Lys replaced by Met)

A new haemoglobin, Hb Helsinki, in which beta 82-Lys (EF6) is replaced by Met, was found in a Finnish family. It was associated with familial erythrocytosis, and the oxygen affinity of the blood was higher than normal. The oxygen equilibrium curves of purified Hb Helsinki and HbA from the same haemolysate have been determined under vaious conditions. "Stripped' Hb Helsinki was found to show normal cooperativity, slightly low oxygen affinity and a reduced Bohr effect at physiological pH. However, the organic phosphates, 2,3-diphosphoglycerate (2,3-DPG) and inositol hexaphosphate (IHP) had a very small effect on Hb Helsinki, and the 2,3-DPG binding constant of deoxygenated Hb Helsinki is close to that of oxyhaemoglobin A. Thus, the replacement of Lys by Met at position 82 dramatically changes the nature of the central cavity of the tetramer and the effect of 2,3-DPG on the respiratory function of the molecule.     

Hb Malm? [beta-97(FG-4)His-->Gln] leading to polycythemia in a Dutch family

We have examined six individuals from a two-generation Dutch family for a suspected hemoglobin (Hb) abnormality. The propositus presented with polycythemia and complained of persistent weakness, headache, and epistaxis. All family members initially showed a normal Hb-electrophoretic pattern, but on isoelectric focusing, three of them displayed a fast-moving band associated with high packed red cell volumes (PCV) and increased red blood cell count. The Hb mutant was analyzed at the DNA level by specific gene fragment amplification (PCR), followed by direct DNA sequencing, and the mutation was confirmed by restriction enzyme analysis. We found a C-->G transversion (CAC-->CAG) at codon 97 of the beta-chain, which corresponded to the His-->Gln amino acid substitution previously described as Hb Malm?. We report here the clinical history of the patient, the effects of phlebotomy treatment, and the effect of subnormal iron conditions on the erythropoietic recovery after phlebotomy. The mechanism responsible for the induction of the higher oxygen affinity is discussed, as are some aspects concerning the occurrence, pathology treatment, and the genetic risk of Hb variants with high O2 affinity.     

Interleukin-1 beta inhibits the intestinal transport of [14C] 3-O-methylglucose in the rat

Interleukin-1 (IL-1) is known to be a hypoglycemic cytokine, but its mechanism of action is still unknown. Since the blood glucose levels depend on the amount of glucose entering and leaving the circulation, this work was conducted to test the hypothesis that the hypoglycemia observed with IL-1beta might result, at least partially, from a reduced intestinal glucose absorption. Male Sprague Dawley rats were injected intraperitoneally (i.p.) with IL-1beta, and a jejunal segment was perfused with [14C] 3-O-methylglucose for 5, 15, 25 and 40 min. Our results showed that IL-1beta significantly inhibited the mucosal uptake of this hexose and reduced its intestinal retention. The time course and the dose response effect for this cytokine were also determined. Studies on the effect of IL-1beta on the activity of the intestinal Na+-K+ ATPase demonstrated a significant inhibition of the pump. The effect of IL-1beta on the hexose transport across the brush border membrane may thus be attributed to its inhibitory effect on the Na+-K+ ATPase.     

Hydrothermal synthesis and crystal structure study of two novel 3-D mellitates {Nd2[C6(COO)6](H2O)6} and {Ho2[C6(COO)6](H2O)6}

Two novel 3-D coordination compounds, Nd2[C6(COO)6](H2O)6(1)and Ho2[C6(COO)6](H2O)6(2), were hydrothermally synthe-sized from mellitic acid and neodymium perchlorate (or holmium perchlorate) in the alkaline aqueous solution and characterized with ele-mental analysis, TG, IR spectrum, and single crystal X-ray diffraction. The two compounds were isostructural and crystallized in the ortho-rhombic system, space group Pnnm, with a=1.3531 (4) nm, b=0.6687 (2) nm, c=1.0224(3) nm, V=0.92523(5) nm3, Z=4, D=2.630 g/cm3, F(000)=696.0, Goof=1.052. Final R indices [I 2Σ(I)]: R1=0.0195, wR2=0.0382 for 1; a=1.3411(2) nm, b=0.6586(1) nm, c=1.0116(2) nm, V=0.8935(3) nm3, Z=4, D=2.877 g/cm3, F(000)=724.0, Goof=1.061. Final R indices [I 2Σ(I)]: R1=0.0200, wR2=0.0479 for 2. In the two compounds 1 and 2, the mellitic acid ligand, in which all the carboxylate groups were deprotonated, had only one kind of coordination mode to bridge metal ions to form four-connected three-dimensional diamondiod networks.     

Role of beta87 Thr in the beta6 Val acceptor site during deoxy Hb S polymerization

Three new Hb S variants containing beta87 Leu, Trp, or Asp instead of Thr were expressed in yeast in order to further define the role of the beta87 position in stability and polymerization of deoxy Hb S. Previous studies showed that hydrophobicity at beta85 Phe and beta88 Leu is critical for stabilization of hemoglobin. Results with the three Hb S beta87 variants, however, showed minimal differences in stability, suggesting that beta87 amino acid hydrophobicity is not critical for stabilization of hemoglobin. Polymerization properties of the variants in the deoxy form, however, were affected by the beta87 amino acid. Polymerization of Hb S beta87 Thr --> Leu and Hb S beta87 Thr --> Trp was preceded by a delay time like Hb S, while Hb S beta87 Thr --> Asp did not show a delay time. In addition, changes in time required for half polymer formation (T1/2) as a function of hemoglobin concentration for Hb S beta87 Thr --> Asp were similar to that for beta87 Thr --> Gln. Hb S beta87 Thr --> Leu polymerized at a lower hemoglobin concentration than Hb S while beta87 Thr --> Trp and Hb S beta87 Thr --> Asp required much higher hemoglobin concentrations for polymer formation. Critical concentration required for deoxy Hb S beta87 Thr --> Asp polymerization was 6- and 2.3-fold greater than that for Hb S beta85 Phe --> Glu and Hb S beta88 Leu --> Glu, respectively. These results suggest that even though beta87 Thr is not a direct interaction site for beta6 Val in deoxy Hb S polymers, it does play a critical role in formation of the hydrophobic acceptor pocket which then promotes protein-protein interactions facilitating formation of stable nuclei and polymers of deoxy Hb S.     

Higher oxygen affinity of sheep Hb C compared to Hb A and Hb B

Sheep which have Hb A, synthesize the perinatal Hb C in response to severe tissue hypoxia. It is known that Hb A displays a higher oxygen affinity than Hb B. The results of this study indicate that Hb C exhibits an oxygen affinity and a Bohr effect higher than those of Hb A and Hb B.     

Synthesis and Crystal Structure of a 4f-3d Complex [La(DMF)_6(H_2O)_3][Cr(CN)_6]·2H_2O

Ｒｅｃｅｎｔｌｙ ,ｔｈｅｒｅｈａｓｂｅｅｎｃｏｎｓｉｄｅｒａｂｌｅｉｎ ｔｅｒｅｓｔｉｎｌａｎｔｈａｎｉｄｅ(Ⅲ )ｈｅｘａｃｙａｎｏｆｅｒｒａｔｅｓａｎｄｔｈｅａｎａｌｏｇｏｕｓｃｏｂａｌｔ(Ⅲ )ａｓｗｅｌｌａｓｃｈｒｏｍｉｕｍ(Ⅲ )ｃｏｍｐｌｅｘｅｓｂｅｃａｕｓｅｏｆｔｈｅｉｒｐｏｔｅｎｔｉａｌａｓｃａｔａｌｙｔｉｃ ,ｓｅｍｉｃｏｎｄｕｃｔｉｖｅ ,ａｎｄｍａｇｎｅｔｉｃｍａｔｅ ｒｉａｌｓ[1～ 4] .Ｆｏｒｅｘａｍｐｌｅ ,ｍａｇｎｅｔｉｃｓｔｕｄｉｅｓｏｎａｓｅｒｉｅｓｏｆｔｈｒｅｅ ｄｉｍｅｎｓｉｏ…     

Synthesis and Structure of a Polymeric Complex [Ce（bphsb）2（NO3）3]n

Polymeric cerium nitrate complex with 1,4-bis (phenylsulfinyl) butane (bphsb) [Ce(bphsb)2(NO3)3]n (1) was synthesized and characterized. The crystal structure of the complex indicated that the ligand bphsb and cerium( Ⅲ ) ion form a polymeric double-bridge chain complex involving 18-membered macrometallocycles. Each cerium ion is coordinated by ten 0 atoms in a distorted 4,4-bicapped square antiprism. In the complex the disulfoxide ligand acts as bis-monodentate O-ligand bridging metal centers.     

Autoradiographic characterization of [3H]inositol (1,4,5) trisphosphate and [3H]inositol (1,3,4,5) tetrakisphosphate binding sites in human brain

Autoradiographic techniques were used to investigate the characteristics of tritiated inositol(1,4,5)trisphosphate ([3H]IP3) and inositol (1,3,4,5) tetrakisphosphate ([3H]IP4) binding to human brain. In brain sections [3H]IP3 exhibited a two-site binding with KD values of 87 nM and 9.3 microM respectively for the higher and lower affinity sites. [3H]IP4 also bound to two sites with KD values of 43 nM and 1.4 microM, respectively. With the conditions fixed in this study, [3H]IP3 and [3H]IP4 autoradiography in the cortex, caudate, hippocampus and cerebellum were performed. The most prominent [3H]IP3 binding among these regions was found in the cerebellum, particularly in the molecular layer. Within the hippocampus, the subiculum and the CA1 region showed much more prominent binding than the other subfields. [3H]IP4, binding was fairly homogeneous in the regions studied, with the exception of a slightly higher binding in the molecular layer of the cerebellum.     

Identification of dihydropyridine (DHP) binding sites on cultured monkey renal cells (GMRC) with a photoaffinity probe (-)-[3H]-azidopine

The low affinity binding sites identified in crude membranes from different excitable tissues with the dihydropyridine (DHP) calcium (Ca2+) channel ligands have confused researches in the field of Ca2+ channels as they can represent low affinity state(s) of the DHP receptor, or they can be labelled with DHP-type Ca2+ channel ligands. The aim of this communication was to provide more evidence for the existence of separate DHP binding sites on the surface of cultured green monkey renal cells (GMRC). The saturation ligand binding experiments with [3H]-nitrendipine (NTP) and photoaffinity labelling studies with (-)-[3H]-azidopine (AZI) were performed in order to identify and further characterize the DHP receptor on cultured GMRC. Specific high affinity sites identified on GMRC with [3H]-NTP (Bmax = 0.78 +/- 0.03 pmol/mg protein and KD = 0.06 +/- 0.1 nmol/l in native cells) and photolabelled with AZI represent DHP receptor on L-type Ca2+ channels. The low affinity binding sites photolabelled with AZI on GMRC (9.84 +/- 2.4 pmol/mg protein and KD = 3.21 +/- 1.25 nmol/l in native cells) were significantly increased after preincubation of GMRC with low concentrations of DHPs nitrendipine and nisoldipine. Preincubation of GMRC with Ca2+ channel agonist (-)BAYK 8644 significantly reduced specific photolabelling with AZI on GMRC and increased low affinity labelling. Preincubation of (+)BAYK 8644 was without any effect. Niguldipine (DHP with the voluminous substituent on the port side of the DHP ring) partially inhibited specific photolabelling with AZI on GMRC and also partially reduced the maximal number of low affinity binding sites labelled with AZI. Our results support the hypothesis of separate subsites in the region of DHP receptor of GMRC and the existence of the "marginal" photolabelling of specific DHP binding sites identified on Ca2+ channels.     

Regulation of IL-6 synthesis in human peripheral blood mononuclear cells by C3a and C3a(desArg)

The anaphylatoxin C3a has been reported to have immunomodulatory effects on a number of different cell types. In this study we investigated the effects of C3a and C3a(desArg) on gene expression and protein secretion of IL-6 in human PBMCs, either alone or in combination with LPS or IL-1beta. C3a or C3a(desArg) alone exhibited no effect on the expression or secretion of IL-6. However, when PBMC were stimulated with LPS or IL-1beta, both C3a and C3a(desArg) were found to enhance IL-6 release by PBMC in a dose-dependent manner. Since C3a has been shown to induce PGE2 production by monocytes, and PGE2 has been shown to influence cytokine production, we investigated the potential role of PGE2 in C3a-mediated enhancement of LPS- and IL-1beta-induced IL-6 production. Indomethacin blocked PGE2 release, but had no influence on the observed effects of C3a, suggesting that the effects of C3a on IL-6 production are independent of PGE2 formation by monocytes. Northern blot analysis showed that C3a as well as C3a(desArg) enhanced LPS-induced mRNA levels for IL-6. Pretreatment of PBMCs with pertussis toxin blocked the functions of C3a and C3a(desArg), indicating that the actions of these two molecules are mediated by a G protein-coupled pathway. Furthermore, we investigated the effects of C3a and C3a(desArg) on induction of NF-kappaB and activating protein-1 binding. Both molecules enhanced LPS-induced NF-kappaB and activating protein-1 binding activity. These results demonstrate the capacity of intact C3a and its circulating des-Arg form to exert immunmodulatory effects in vitro.     

Mutation in the thyroid hormone receptor beta gene (A317T) in a Thai subject with resistance to thyroid hormone

Analysis of the thyroid hormone receptor beta (TRbeta) gene of a Thai female with the syndrome of resistance to thyroid hormone (RTH) revealed a missense mutation at codon 317, changing the guanine in nucleotide 1234 to an adenine that results in the replacement of the normal alanine (GCT) with a threonine (ACT). The proposita was heterozygous, and this mutation was not present in her parents and her sister, compatible with a neomutation. This is the first report of TRbeta gene mutation causing RTH in an individual of Thai origin.