CD4+ CD25+ Treg cells inhibit human memory gammadelta T cells to produce IFN-gamma in response to M tuberculosis antigen ESAT-6

T cells play an important role in innate immunity against infections; however, the regulation of these cells remains largely unknown. In the present study, we show that ESAT-6, an antigen of Mycobacterium tuberculosis, induces IFN- secretion by human T cells. In addition, ESAT-6 also induces the activation and proliferation of T cells. Phenotypic analysis indicates that IFN-–producing T cells are mainly effector memory cells with the surface phenotype of CD45RA^–CD62L^–CCR7^–. These results were further confirmed by the fact that naive T cells from cord blood did not produce IFN- in response to ESAT-6. Further studies indicated that stimulation with ESAT-6 directly induced purified T cells to produce IFN-, independent of both antigen-presenting cells and CD4⁺ T cells. Unexpectedly, depletion of CD4⁺ T cells markedly enhanced IFN- production by T cells, indicating that CD4⁺ T cells regulate the response of T cells. Importantly, CD4⁺CD25⁺ T regulatory (Treg) cells but not CD4⁺CD25^– T cells significantly inhibited IFN- production by T cells. Taken together, these data demonstrate for the first time that Treg cells can play an important role in the regulation of immune responses of antigen-specific human memory T cells.     

Adult murine hematopoiesis can proceed without beta1 and beta7 integrins

The function of 41 and 47 integrins in hematopoiesis is controversial. While some experimental evidence suggests a crucial role for these integrins in retention and expansion of progenitor cells and lymphopoiesis, others report a less important role in hematopoiesis. Using mice with a deletion of the 1 and the 7 integrin genes restricted to the hematopoietic system we show here that 41 and 47 integrins are not essential for differentiation of lymphocytes or myelocytes. However, 17 mutant mice displayed a transient increase of colony-forming unit (CFU-C) progenitors in the bone marrow and, after phenylhydrazine-induced anemia, a decreased number of splenic erythroid colony-forming units in culture (CFUe's). Array gene expression analysis of CD4⁺CD8⁺ double-positive (DP) and CD4^–CD8^– double-negative (DN) thymocytes and CD19⁺ and CD4⁺ splenocytes did not provide any evidence for a compensatory mechanism explaining the mild phenotype. These data show that 41 and 47 are not required for blood cell differentiation, although in their absence alterations in numbers and distribution of progenitor cells were observed.      

The Interaction of Hemoglobin E With beta Thalassemia: A Study of Hemoglobin Synthesis in a Family of Mixed Burmese and Iranian Origin

A 5-yr-old girl with hemoglobin E- thalassemia was discovered in a family of mixedorigin. The father is Iranian (-thalassemiatrait) and the mother is Burmese (hemoglobin-E trait). Hemoglobin synthesis wasstudied in vitro in the blood of theproposita and family members. In the subjects with hemoglobin E trait the ratio ofthe quantity of hemoglobin A to hemoglobin E was 3:1. However. the ^A/^Esynthesis ratio in reticulocytes was in therange of 1.5-2.18, and the specific activityof ^E was 31%-49% greater than ^A, suggesting instability of hemoglobin E withpreferential destruction of abnormal hemoglobin. The blood of the proposita exhibitedonly hemoglobin F and hemoglobin E andreticulocytes and bone marrow showed no^A synthesis. This Iranian -thalassemiagene is therefore of the ° type. The ^E/synthesis ratio (approximately 0.74) inblood of the proposita was similar to the^A/ ratio in mildly affected relatives withthalassemia trait. These results suggestthat the severity of the hemoglobin E-thalassemia syndrome is attributable toboth instability and defective synthesis ofhemoglobin E in association with absent^A synthesis due to a ° thalassemia gene.

Submitted on March 19, 1973 Revised on May 4, 1973 Accepted on May 8, 1973     

Variable protection of beta 3-integrin--deficient mice from thrombosis initiated by different mechanisms

Platelet integrin IIb3 (GPIIb/IIIa) plays a central role inthe initiation of arterial thrombosis, but its contribution todisseminated microvascular thrombosis is less well defined. Therefore,wild-type mice (3^+/+), 3-integrin-deficient mice(3^/), and wild-type mice treated with a hamstermonoclonal antibody (1B5) that blocks murine IIb3 function weretested in models of large-vessel and microvascular thrombosis. In thelarge-vessel model, ferric chloride was used to injure the carotidartery, and the time to thrombosis was measured. In 3^+/+mice, the median time to occlusion was 6.7 minutes, whereas occlusion did not occur in any of the 3^/ mice tested(P < .001). Fab and F(ab')₂ fragments of1B5 increased the median time to occlusion. To initiate systemicintravascular thrombosis, prothrombotic agents were administeredintravenously, and platelet thrombus formation was monitored by thedecrease in circulating platelet count. Three minutes after theinjection of adenosine diphosphate (ADP), collagen + epinephrine,or tissue factor, the platelet counts in 3^+/+ micedecreased by 289, 424, and 429 × 10³/µL, respectively.3^/ mice and wild-type mice pretreated with 1B5 Fab(1 mg/kg, IP) were nearly completely protected from the effects of ADP.In contrast, 3^/ mice were only partially protectedfrom the effects of collagen + epinephrine and minimally protectedfrom the effects of tissue factor. In all cases, less fibrin becamedeposited in the lungs of 3^/ mice than in wild-typemice. These results suggest that though IIb3 plays adominant role in large-vessel thrombosis, it plays a variable role insystemic intravascular thrombosis.     

Targeting NF-kappaB in Waldenstrom macroglobulinemia

The nuclear factor-B (NF-B) path-way has been implicated in tumor B-cell survival, growth, and resistance to therapy. Because tumor cells overcome single-agent antitumor activity, we hypothesized that combination of agents that target differentially NF-B pathway will induce significant cytotoxicity. Therapeutic agents that target proteasome and Akt pathways should induce significant activity in B-cell malignancies as both pathways impact NF-B activity. We demonstrated that perifosine and bortezomib both targeted NF-B through its recruitment to the promoter of its target gene IB using chromatin immunoprecipitation assay. This combination led to synergistic cytotoxicity in Waldenstrom macroglobulinemia (WM) cells that was mediated through a combined reduction of the PI3K/Akt and ERK signaling pathways, found to be critical for survival of WM cells. Moreover, a combination of these drugs with the CD20 monoclonal antibody rituximab further increased their cytotoxic activity. Thus, effective WM therapy may require combination regimens targeting the NF-B pathway.      

A310 helical turn is essential for the proliferation-inhibiting properties of macrophage inflammatory protein-1 alpha (CCL3)

Despite possessing marked structural similarities, the chemokines macrophage inflammatory protein-1 (MIP-1; CCL3) and RANTES (CCL5) display differential activity in hematopoietic progenitor-cell-inhibitory assays, with MIP-1 being active and RANTES inactive in this context. We have sought to identify the key structural determinants of this property of MIP-1. This has involved constructing MIP-1/RANTES chimeras by swapping structural domains between the 2 proteins. Results indicate that, in contrast to other chemokine functions, neither the N nor the C termini are key determinants of inhibitory activity. The motif that appears to be most important for this activity lies between the second and fourth cysteines of MIP-1 and further domain swap analysis has narrowed this down to the 3₁₀ helical turn preceding the first -strand in MIP-1. More detailed analysis has highlighted the role played by a specific dipeptide motif in the proliferation-inhibitory activity of chemokines. The involvement of the 3₁₀ helical-turn motif in chemokine function is unprecedented and this study therefore identifies a novel, functionally essential motif within chemokines. In addition, this study further attests to the alternative mechanisms of action used by MIP-1 in inhibition of hematopoietic progenitor-cell proliferation and regulation of leukocyte migration.      

Coinduction of Embryonic and Adult-Type Globin mRNAs by Sodium Butyrate and Trichostatin A in Two Murine Interleukin-3-Dependent Bone Marrow-Derived Cell Lines

Using an RNase protection assay, globin mRNA species expressed inclones derived from Ba/F3 and B6SUtA cells transfected with theerythropoietin receptor (EpoR) and selected with erythropoietin (Epo)were compared with globin mRNA species induced in corresponding parental cells by sodium butyrate (SB) and trichostatin A (TSA). ^Major/^minor- and -1/-2-globinmRNAs were the major species, with trace amounts of -globin mRNA,formed in Epo-stimulated EpoR⁺ Ba/F3 clones, whereas SBand TSA allowed expression of all species of globin mRNAs, ie, ,h1, ^major/^minor, , and -1/-2,in parental Ba/F3 cells. In contrast, - and -1/-2-globinmRNAs were the major species present in Epo-stimulated EpoR⁺ B6SUtA clones, whereas SB and TSA activated -,h1-, ^S/^T-, and -1/-2-globingenes in parental B6SUtA cells; -globin mRNA was not detected in SB-and TSA-treated B6SUtA cells. Because TSA is a specific inhibitor ofhistone deacetylase, the mimicry of action exhibited by SB and TSAsuggests that the effects of SB are mediated through its ability toinhibit histone deacetylase and that histone deacetylase is an integralpart of the repression of globin genes in theseinterleukin-3-dependent cells. Efficient coinduction of embryonic andadult types of globin mRNA in bone marrow cell lines derived from adultmice indicates that adult hematopoietic precursors possess an embryonicnature. These cell lines are useful models to study the mechanism(s) ofdevelopmental globin gene switching.     

Synthesis of Hemoglobin Abraham Lincoln (beta 32 leu rarr pro)

Globin chain synthesis was studied in vitrowith reticulocytes from a patient heterozygous for Hb Abraham Lincoln, an unstable beta chain variant. Synthesis of-chains by the reticulocytes exceededtotal -chain synthesis, and a substantialfraction (about 16%) of radioactivityincorporated into globin was recoveredfrom the cells as uncombined subunits.In a time-course study, the ratio of : -chain specific activity was found to increase progressively in a nearly linearmanner, suggesting that a fraction ofnewly synthesized -chains had undergone rapid destruction. The specific activity of the abnormal -chain was nearlythree times that of ^A. The rate of synthesis of the -chain of Hb AbrahamLincoln appeared to be approximately halfthat of the -chain of Hb A.

Submitted on September 13, 1973 Revised on November 1, 1973 Accepted on November 6, 1973     

Modulation of angiogenesis by omega-3 polyunsaturated fatty acids is mediated by cyclooxygenases

The potential role of dietary fats in cancer is attracting considerable interest within the community. Both epidemiologic and experimental findings suggest that omega-3 polyunsaturated fatty acids (-3 PUFAs), which are almost absent from typical Western diets, exert protective effects against cancer progression, although the precise mechanism of this suppression remains unknown. One of the potential targets for -3 PUFAs in cancer suppression is angiogenesis, a process of new blood vessel formation within rapidly growing tumors. Here, we demonstrate that -6 PUFAs stimulate and -3 PUFAs inhibit major proangiogenic processes in human endothelial cells, including the induction of angiopoietin-2 (Ang2) and matrix metalloprotease-9, endothelial invasion, and tube formation, that are usually activated by the major -6 PUFA arachidonic acid. The cyclooxygenase (COX)–mediated conversion of PUFAs to prostanoid derivatives participated in modulation of the expression of Ang2. Thus, the -6 PUFA–derived prostaglandin E2 augmented, whereas the -3 PUFA–derived prostaglandin E3 suppressed the induction of Ang2 by growth factors. Our findings are consistent with the suggestion that PUFAs undergo biotransformation by COX-2 to lipid mediators that modulate tumor angiogenesis, which provides new insight into the beneficial effects of -3 PUFAs.      

Regulation of tumor necrosis factor receptor-1 and the IKK-NF-kappaB pathway by LDL receptor-related protein explains the antiinflammatory activity of this receptor

Low-density lipoprotein receptor–related protein (LRP-1) functions in endocytosis and in cell signaling directly (by binding signaling adaptor proteins) or indirectly (by regulating levels of other cell-surface receptors). Because recent studies in rodents suggest that LRP-1 inhibits inflammation, we conducted activity-based protein profiling experiments to discover novel proteases, involved in inflammation, that are regulated by LRP-1. We found that activated complement proteases accumulate at increased levels when LRP-1 is absent. Although LRP-1 functions as an endocytic receptor for C1r and C1s, complement protease mRNA expression was increased in LRP-1–deficient cells, as was expression of inducible nitric oxide synthase (iNOS) and interleukin-6. Regulation of expression of inflammatory mediators was explained by the ability of LRP-1 to suppress basal cell signaling through the IB kinase–nuclear factor-B (NF-B) pathway. LRP-1–deficient macrophages, isolated from mice, demonstrated increased expression of iNOS, C1r, and monocyte chemoattractant protein-1 (MCP-1); MCP-1 expression was inhibited by NF-B antagonism. The mechanism by which LRP-1 inhibits NF-B activity involves down-regulating cell-surface tumor necrosis factor receptor-1 (TNFR1) and thus, inhibition of autocrine TNFR1-initiated cell signaling. TNF-–neutralizing antibody inhibited NF-B activity selectively in LRP-1–deficient cells. We propose that LRP-1 suppresses expression of inflammatory mediators indirectly, by regulating TNFR1-dependent cell signaling through the IB kinase–NF-B pathway.      

A functional 14-3-3zeta-independent association of PI3-kinase with glycoprotein Ib alpha, the major ligand-binding subunit of the platelet glycoprotein Ib-IX-V complex

Engagement of the adhesion receptor glycoprotein (GP) Ib-IX-V by von Willebrand factor (VWF) mediates platelet adhesion to damaged vessels and triggers platelet activation and thrombus formation in heart attack and stroke. GPIb-IX-V contains distinct 14-3-3–binding sites at the GPIb C-terminus involving phosphorylation of Ser609, an upstream site involving phosphorylated Ser587/Ser590, and a protein kinase A (PKA)–dependent site on GPIbβ involving Ser166. 14-3-3 regulates the VWF-binding affinity of GPIb-IX-V and inhibiting 14-3-3 association blocks receptor signaling, suggesting a key functional role for 14-3-3. We used deletion mutants of GPIb expressed in Chinese hamster ovary (CHO) cells to define the relationship of 14-3-3 binding to another GPIb-IX-V–associated signaling protein, phosphoinositide 3-kinase (PI3-kinase). Pull-down experiments involving glutathione S-transferase (GST)–PI3-kinase/p85-subunit and GST–14-3-3 indicated that both proteins interacted with contiguous GPIb sequences 580 to 590/591 to 610. Deleting these, but not upstream sequences of GPIb expressed in CHO cells, inhibited VWF/ristocetin-dependent Akt phosphorylation, relative to wild-type receptor, confirming this region encompassed a functional PI3-kinase–binding site. Pull-down experiments with GST-p85 truncates indicated the GPIb-binding region involved the p85 breakpoint cluster region (BCR) domain, containing RSXSXP. However, pull-down of GPIb-IX was unaltered by mutation/deletion/phosphorylation of this potential 14-3-3–binding sequence in mutant constructs of GST-p85, suggesting PI3-kinase bound GPIb independently of 14-3-3; 14-3-3 inhibitor peptide R18 also blocked pull-down of receptor by GST-14-3-3 but not GST-p85, and GST-p85 pull-downs were unaffected by excess 14-3-3. Together, these data suggest the GPIb C-terminus regulates signaling through independent association of 14-3-3 and PI3-kinase.     

Involvement of Na+/Ca2+ Exchanger in Inside-Out Signaling Through the Platelet Integrin alpha IIbbeta 3

The platelet integrin _IIb3 has becomea new target for the treatment of pathological thrombosis. It becomesapparent that the affinity of _IIb3 forits ligands is dynamically regulated by inside-out signaling. However,the components that couple diverse intracellular signals to thecytoplasmic domains of _IIb3 remain obscure. Employing a chymotrypsin-induced_IIb3 activation model, we previouslyproposed the hypothesis that Na⁺/Ca^{2 +}exchanger (NCX) may be involved in inside-out signaling (Shiraga et al:Blood 88:2594, 1996). In the present study, employing two unrelated Na⁺/Ca²⁺ exchange inhibitors,3,4-dichlorobenzamil (DCB) and bepridil, we investigatedthe role of NCX in platelet activation induced by various agonists indetail. Both inhibitors abolished platelet aggregation induced by allagonists examined via the inhibition of_IIb3 activation. Moreover, theseinhibitors abolished _IIb3 activationinduced by phorbol 12-myristate 13-acetate or A23187. On the otherhand, neither of these inhibitors showed apparent inhibitory effects onprotein phosphorylation of pleckstrin or myosin light chain, or anincrease in intracellular calcium ion concentrations evoked by 0.1 U/mLthrombin. These effects of the NCX inhibitors are in striking contrastto those of protein kinase C inhibitor, Ro31-8220. Biochemical andultrastructural analyses showed that NCX inhibitors, particularly DCB,made platelets "thrombasthenic". These findings suggest that theNCX is involved in the common steps of inside-out signaling throughintegrin _IIb3.     

Association Between Ligand-Induced Conformational Changes of Integrin alpha IIbbeta 3 and alpha IIbbeta 3-Mediated Intracellular Ca2+ Signaling

Platelet _IIb3 is a prototypic integrinand plays a critical role in platelet aggregation. Occupancy of_IIb3 with multivalent RGD ligands, suchas fibrinogen, induces both expression of ligand-induced binding sites(LIBS) and _IIb3 clustering, which arethought to be necessary for outside-in signaling. However, theassociation between LIBS expression and outside-in signaling remainselusive. In this study, we used various_IIb3-specific peptidomimetic compounds asa monovalent ligand instead of fibrinogen and examined the associationbetween LIBS expression and outside-in signaling such as_IIb3-mediated intracellularCa²⁺ signaling. Using a set of monoclonal antibodies(MoAbs) against LIBS, we showed that antagonists can be divided intotwo groups. In group I, antagonists can induce LIBS on both_IIb and ₃ subunits. In group II,antagonists can induce LIBS on the _IIb subunit, but noton the ₃ subunit. Inhibition studies suggested thatgroup I and group II antagonists interact with distinct but mutually exclusive sites on _IIb3. Neither group Inor group II antagonist increased intracellular Ca²⁺concentrations ([Ca²⁺]_i) in nonactivatedplatelets. All antagonists at nanomolar concentrations abolished theincrease in [Ca²⁺]_i in 0.03 U/mLthrombin-stimulated platelets, which is dependent on bothfibrinogen-binding to _IIb3 andplatelet-aggregation. However, only group I antagonists at higherconcentrations dose-dependently augmented the[Ca²⁺]_i increase, which is due toaggregation-independent thromboxane A₂ production. Thisincrease in [Ca²⁺]_i was not observed inthrombasthenic platelets, which express no detectable_IIb3. Thus, only the group I antagonists,albeit a monovalent ligand, can initiate_IIb3-mediated intracellular Ca²⁺ signaling in the presence of thrombin stimulation.Our findings strongly suggest the association between ₃LIBS expression and _IIb3-mediatedintracellular Ca²⁺ signaling in platelets.     

Presentation of ovalbumin internalized via the immunoglobulin-A Fc receptor is enhanced through Fc receptor gamma-chain signaling

The mechanism of enhanced presentation of ovalbumin (OVA)internalized as immunoglobulin A (IgA)-OVA via the IgA Fc receptor (FcR) was analyzed by focusing on the role of the FcR-associated  chain. Comparison of B-cell transfectants expressing FcR plus wild-type (WT)  chain or  chain in which theimmunoreceptor tyrosine-based activation motif (ITAM) was altered bytyrosine mutation or substitution with the ITAM of FcRIIA showedthat signaling-competent ITAM was not required for endocytosis ofIgA-OVA. However, antigen presentation was impaired by ITAM changes.Signaling-competent -chain ITAM appeared necessary for transport ofligated FcR to a lamp-1⁺ late endocytic compartment forremodeling and/or activation of that compartment and also for efficientdegradation of IgA complexes. Moreover, FcR ligation also activatedefficient processing of nonreceptor-targeted antigen. Theresults suggest that -chain signaling activates the antigenprocessing compartment.     

Plasma infusions in immunologic deficiency states: metabolic and therapeutic studies

Serial immunologic measurements were used to study the metabolic behavior of the immune globulins (_G-, _M- and _A-globulins) in patients with agammaglobulinemia after plasmapheresis and plasma infusion and in newborninfants after exchange transfusion. These studies were supplemented by metabolic and distribution studies of I¹³¹-labeled _A-globulin (isolated from serumor breast milk) and I¹³¹-labeled _G-globulin in normal and agammaglobulinemic subjects. The therapeutic benefit of periodic plasma infusions in patientswith agammaglobulinemia and the Aldrich syndrome was also assessed.

In the agammaglobulinemic patients, the mean half-lives of _G-, _M- and_A-globulin were 32, 9.6 and 5.9 days, respectively. In the transfused infants,the mean half-lives of _M- and _A-globulins were 7.4 and 4.3 days, respectively.Agreement existed between simultaneously determined immunologic and radioactive survival times, except when I¹³¹-labeled _A-globulin isolated fromserum was used; this preparation had a shorter half-life than the _A-globulinof infused plasma, probably as a result of denaturation during the isolationprocedure. Studies on 2 normal and 3 agammaglobulinemic subjects showedthat 65 to 85 per cent of breast milk I¹³¹-labeled _A-globulin was distributedwithin the tissues. I¹³¹-labeled _A-globulin was not demonstrable in the breastmilk of 2 lactating women or in the saliva of 2 normal subjects. No _A-globulincould be demonstrated in the saliva of an agammaglobulinemic patient afterplasma infusion which raised the serum _A-globulin concentration to 50mg./100 ml.

The use of plasma instead of commercial -globulin for the therapy of immunologic deficiency states has several advantages. Plasma contains all threeimmune globulins, provides greater quantities of _G-globulin than can begiven by intramuscular injections, and is more acceptable to the patient. Because of the risk of serum hepatitis, this mode of therapy in the routine management of agammaglobulinemia is endorsed only if special precautions aretaken. A therapeutic trial of plasma infusions in 2 patients with the Aldrichsyndrome gave promising results.

Submitted on January 7, 1966 Accepted on April 25, 1966     

Bone Marrow and Peripheral Blood Globin Synthesis in an American Black Family With Beta Thalassemia

Synthesis of globin chains in bonemarrow and peripheral blood samplesfrom a black family with mild betathalassemia was compared with similar studies in white people. Blood andbone marrow were incubated with ¹⁴C-leucine, globin chains were isolated,and / and / ratios were calculated.The results of studies of globin synthesis in homozygotes of differentraces were similar, despite the differences in severity of clinical disease. Inthe heterozygotes, there was a significant defect in beta synthesis in theperipheral blood of white subjects,while in two of three black patients the/ ratio was in the normal range. Although there was no evidence of segregation of an alpha thalassemia genein this black family to explain the unusual / ratios, the presence of sucha gene in the heterozygotes could notbe excluded.

Submitted on September 2, 1971 Revised on November 5, 1971 Accepted on December 15, 1971     

Globin Messenger RNA in Hemoglobin H Disease

Functional messenger RNA (mRNA) forhuman globin synthesis was isolated fromreticulocytes of each of two patients withhemoglobin H disease. The RNA wastested for its capacity to direct globinsynthesis in a messenger RNA-dependentcell-free system derived from Krebs Type IImouse ascites tumor cells. In each case,hemoglobin H disease mRNA directed thesynthesis of a great excess of -globinchains relative to -globin chains of hemoglobin A. The / synthetic ratios obtained in the cell-free system at saturatingconcentrations of mRNA were >22 and>15, respectively, for the two hemoglobinH disease mRNA preparations, whereasthe / synthetic ratios obtained by incubation of intact reticulocytes from thesesame patients were 2.6 and 2.8, respectively. The / synthetic ratio obtained inthe cell-free system did not vary whenlower concentrations of hemoglobin Hdisease mRNA were used. A marked decrease in the amount of functional -globin-chain mRNA relative to -chainmRNA is therefore associated with the decreased -chain synthesis observed inhemoglobin H disease. This decrease in-chain-specific mRNA activity is greaterthan expected from the / synthetic ratioof intact reticulocytes in hemoglobin Hdisease.

Submitted on May 1, 1973 Revised on June 15, 1973 Accepted on June 16, 1973     

Hemoglobin Rush [beta101 (G3) Glutamine]: A New Unstable Hemoglobin Causing Mild Hemolytic Anemia

A mild hemolytic anemia in a 43-yr-old blackwoman was attributed to the presence of an abnormal hemoglobin (Hb Rush) which migratedcathodically to Hb A at pH 8.0. Its structuralabnormality was found to be in the -chain,101 (G3) glu gln. Another electrophoreticband at pH 8.0 proved to be a hybrid tetramer(^A₂^ARush). Hb Rush is heat unstable. Alikely explanation of the instability is thepresence of an uncovered positive charge in thecentral cavity where normally glutamic acid inposition 101 neutralizes arginine in position104 contributing to the net neutrality in thisregion. This neutrality is disturbed by the substitution of glutamic acid by glutamine in HbRush.

Submitted on May 10, 1973 Revised on June 25, 1973 Accepted on July 16, 1973     

Novel exon 12 mutations in the HIF2A gene associated with erythrocytosis

Erythrocytosis can arise from deregulation of the erythropoietin (Epo) axis resulting from defects in the oxygen-sensing pathway. Epo synthesis is controlled by the hypoxia inducible factor (HIF) complex, composed of an and a β subunit. There are 2 main subunits, HIF-1 and HIF-2. Recently, a HIF-2 Gly537Trp mutation was identified in a family with erythrocytosis. This raises the possibility of HIF2A mutations being associated with other cases of erythrocytosis. We now report a subsequent analysis of HIF2A in a cohort of 75 erythrocytosis patients and identify 4 additional patients with novel heterozygous Met535Val and Gly537Arg mutations. All patients presented at a young age with elevated serum Epo. Mutations at Gly-537 account for 4 of 5 HIF2A mutations associated with erythrocytosis. These findings support the importance of HIF-2 in human Epo regulation and warrant investigation of HIF2A in patients with unexplained erythrocytosis.      

The Characterization of Hemoglobin Shimonoseki

Hemoglobin Shimonoseki, discovered in western Japan in 1960, has beenfurther characterized as a mutant with abnormal -polypeptide chains on thebasis of:

(1) The presence of a minor hemoglobin component migrating cathodallyat pH 8.6 to Hb A₂, presumably ₂^Sh₂^A₂.

(2) Hybridization studies.

(3) Fingerprinting of isolated -polypeptide chains.

Hemoglobin Sh is characterized by the substitution of arginine for glutamine at residue 54 and can therefore be designated as ₂^{54 Arg}₂^A.

Submitted on December 17, 1963 Accepted on March 7, 1964