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C T Noguchi

Publications and source records attributed to C T Noguchi.

At least 55 records · Page 3Linked to original sources
PubMed1991

Activation of globin gene expression by cDNAs from induced K562 cells. Evidence for involvement of ferritin in globin gene expression.

We have isolated non-globin cDNA clones specific for erythroid differentiation from K562 human erythroleukemia cells and have identified those that may regulate globin gene transcription. A cDNA library was constructed from K562 cells induced by hemin for production of embryonic and fetal hemoglobins and screened against cDNA from uninduced K562 cells. Full-length clones specific for induced K562 cells were ligated into a eukaryotic expression vector and transfected into HeLa cells to allow for production of the corresponding coded polypeptide. The ability to increase epsilon- or gamma-globin promoter activity was identified using cotransfection with a second vector containing a globin gene promoter fused to a reporter gene. Six of the induced K562-specific clones exhibited the ability to increase the levels of the reporter genes, bacterial chloramphenicol acetyltransferase and human growth hormone. Sequencing analyses of these clones indicated that five were homologous to ferritin heavy and light chains and one had no homology with known DNA or protein sequences. The ferritin light chain cDNA had the greatest effect on globin gene promoter activation, increasing the gamma-globin promoter activity by 6-8-fold. The activation of the globin gene promoter in the absence of globin gene translation suggests that ferritin (or iron) may have a direct role in globin gene transcription. The subtractive library cloning strategy has enabled us to isolate cDNA clones that activate specific gene promoter without the requirement of direct DNA binding. This approach may allow further identification of the genes encoding proteins that are involved in the control of erythropoiesis.

Base Sequence↗
PubMed1991

Hemoglobin aggregation and pseudosickling in vitro of hemoglobin Setif-containing erythrocytes.

Erythrocytes from individuals heterozygous for hemoglobin Setif (alpha 94 Asp----Tyr) sickle in vitro without deoxygenation when incubated in chloride buffer due to hemoglobin aggregation. We now report quantitative studies of hemoglobin polymerization and deformability in these cells. Hemoglobin polymer gradually increased in intact cells during a 24 h incubation period at 24 degrees C. After 24 hr, about 80% of the cells in 290 mOsm sodium chloride buffer contained polymer which appeared as short rods compared to greater than 99% containing polymer at 450 mOsm. Similar proportions of cells were morphologically sickled. Deformability of erythrocytes with 40% hemoglobin Setif incubated in 290 mOsm buffer at 37 degrees C decreased to 80% of normal by 210 min but in 450 mOsm decreased to 50% after only 30 min as measured by the ektacytometer. However, at 4 degrees C deformability remained normal even in 450 mOsm buffer. The solubility of gelled hemolysate containing 40% hemoglobin Setif was 24 g/dl and 21 g/dl at 290 and 459 mOsm buffer respectively. The gel persisted at 4 degrees C with a solubility of 26 g/dl, but melted when dialyzed into sodium phosphate or potassium phosphate buffer. These data suggest that hemoglobin polymerization, reduced deformability, and sickling of hemoglobin Setif-containing erythrocytes are related to reduced hemoglobin solubility. The rate and extent of intracellular polymerization in vitro are considerably reduced (as in the case of sickle trait) compared with erythrocytes from individuals with sickle cell anemia. Hence, the slower kinetics of hemoglobin aggregation in hemoglobin Setif-containing cells provide an alternate system for studying hemoglobin polymerization and abnormal rheology.

Anemia, Sickle Cell↗
PubMed1991

Effects of alpha-thalassemia and sickle polymerization tendency on the urine-concentrating defect of individuals with sickle cell trait.

A defect in urine concentrating ability occurs in individuals with sickle cell trait (HbAS). This may result from intracellular polymerization of sickle hemoglobin (HbS) in erythrocytes, leading to microvascular occlusion, in the vasa recta of the renal medulla. To test the hypothesis that the severity of the concentrating defect is related to the percentage of sickle hemoglobin present in erythrocytes, urinary concentrating ability was examined after overnight water deprivation, and intranasal desmopressin acetate (dDAVP) in 27 individuals with HbAS. The HbAS individuals were separated into those who had a normal alpha-globin genotype (alpha alpha/alpha alpha), and those who were either heterozygous (-alpha/alpha alpha) or homozygous (-alpha/-alpha) for gene-deletion alpha-thalassemia, because alpha-thalassemia modulates the HbS concentration in HbAS. The urinary concentrating ability was less in the alpha alpha/alpha alpha genotype than in the -alpha/alpha alpha or -alpha/-alpha genotypes (P less than 0.05). After dDAVP, the urine osmolality was greater in patients with the -alpha/-alpha genotype than with the -alpha/alpha alpha genotype (882 +/- 37 vs. 672 +/- 38 mOsm/kg H2O) (P less than 0.05); patients with the -alpha/alpha alpha genotype had greater concentrating ability than individuals with a normal alpha-globin gene arrangement. There was an inverse linear correlation between urinary osmolality after dDAVP and the percentage HbS in all patients studied (r = -0.654; P less than 0.05). A linear correlation also existed for urine concentrating ability and the calculated polymerization tendencies for an oxygen saturation of 0.4 and O (r = -0.62 and 0.69, respectively). We conclude that the severity of hyposthenuria in HbAS is heterogeneous. It is determined by the amount of HbS polymer, that in turn is dependent upon the percentage HbS, which is itself related to the alpha-globin genotype.

Adult↗
PubMed1990

Hematologic responses of patients with sickle cell disease to treatment with hydroxyurea.

Because fetal hemoglobin contains gammaglobin chains instead of beta chains, it is not affected by the genetic defect that causes sickle cell disease. Increased levels of fetal hemoglobin decrease the tendency toward intracellular polymerization of sickle hemoglobin that characterizes this disease. Hydroxyurea is one of several cytostatic agents that have been shown to increase the production of fetal hemoglobin in some patients with sickle cell disease. We studied the effects of hydroxyurea administration in 10 hospitalized patients with sickle cell disease, each of whom was treated for three months. Seven patients responded with a 2- to 10-fold increase in fetal hemoglobin, from a mean (+/- SD) of 1.6 +/- 1.6 percent of total hemoglobin to 6.8 +/- 4.7 percent; three patients had fetal-hemoglobin levels of 10 to 15 percent of total hemoglobin. Three did not respond to treatment. Four of the patients who responded were retreated with hydroxyurea after one to four months without treatment and were found to have larger increases in fetal-hemoglobin levels. In most patients, levels were still rising at the end of the study, even after 90 days of therapy. Fetal-hemoglobin levels tended to peak at dosages of hydroxyurea that were myelosuppressive. In the patients who responded to treatment, there were significant increases in the percentage of reticulocytes and erythrocytes containing fetal hemoglobin and in the amount of fetal hemoglobin within these cells. The percentage of dense red cells decreased in the patients who responded to treatment. The tendency toward intracellular polymerization at physiologic oxygen saturation was reduced by about 33 percent in the cells containing fetal hemoglobin, whereas there was no change in the other cells. We conclude that hydroxyurea is effective in increasing the production of fetal hemoglobin, which in this study was found to be associated with a small decrease in hemolysis and an increase in hemoglobin levels despite myelosuppression. Controlled, prospective trials are necessary to establish whether these effects will lead to clinical benefit.

Adult↗
PubMed1990

Microcirculatory adaptations in sickle cell anemia: reactive hyperemia response.

With the technique of laser-Doppler velocimetry, cutaneous blood flows in the forearm of patients with stable sickle cell disease after graded periods of proximal ischemia were compared with normal subjects matched for age, race, and sex, and with patients with anemia caused by beta(+)-thalassemia. In the sickle cell patients the reactive hyperemia was characterized by an increased time interval between the release of the occlusion and the peak amplitude response (time-to-peak) and by a greater period of blood flow above the base-line value (payback ratio) compared with controls. In addition, prolongation of the occlusion period led to an augmentation in the magnitude of the characteristic basal flow oscillations or an induction of this phenomenon at sites not exhibiting it before ischemia. Base-line or ischemia-provoked flow oscillations of either this magnitude or frequency were only observed in normal or thalassemic controls during brief intervals in the rapidly decaying portion of the hyperemic response and in one subject with homozygous hemoglobin C disease. These results would support a model of a local integrative control of microcirculatory blood flow, which appears to become augmented, synchronized, and sustained in sickle cell subjects.

Adaptation, Physiological↗
PubMed1989

Effect of polymerization tendency on haematological, rheological and clinical parameters in sickle cell anaemia.

The polymerization tendency of sickle haemoglobin was estimated as a function of oxygen saturation in 30 patients with homozygous sickle cell anaemia. The deformability of their erythrocytes was also measured, by initial-flow-rate filtration at 37 degrees C through pores of 5 microns diameter, and clinical severity was assessed using a visual analogue scale. By means of partial correlation analysis, it was found that correlations between haematological, rheological, and clinical parameters in sickle cell anaemia could be explained on the basis of an association of each variable with polymerization tendency. Patients with the greatest tendency to form polymer had the least deformable erythrocytes and perceived their disease to be more severe as judged by the visual analogue scale. Polymer formation also appeared to be a determinant of the number of dense cells which, in turn, determine haemolytic rate and erythrocyte deformability.

Anemia, Sickle Cell↗
PubMed1989

Erythrocyte heterogeneity in sickle cell disease: effect of deoxygenation on intracellular polymer formation and rheology of sub-populations.

Erythrocytes from 12 patients with homozygous sickle cell disease in the steady state were fractionated on a Percoll-Stractan density gradient. Erythrocyte deformability was measured by initial-flow-rate filtration through pores of 5 microns diameter and erythrocyte polymer content was calculated as a function of oxygen saturation. Density fractionated sub-populations of sickle cells showed distinct rheological characteristics, the filterability of dense cells being impaired by minimal oxygen desaturation with the apparent formation of little or no intracellular polymer. Lighter cell fractions required a greater degree of deoxygenation and polymer formation to impair deformability, although this occurred prior to morphological sickling. Dense cells therefore exert a disproportionate effect on blood rheology in sickle cell disease and are likely to have an adverse rheological effect in vivo at arterial oxygen tension.

Anemia, Sickle Cell↗
PubMed1989

Intracellular polymerization. Disease severity and therapeutic predictions.

The extent of intracellular polymerization of hemoglobin S, leading to loss of erythrocyte deformability and eventual morphological sickling, is primarily determined by oxygen saturation and intracellular hemoglobin concentration and composition. Epidemiological analysis of sickle cell disease severity among the sickle syndromes and studies of the biophysics of intracellular polymerization were used to estimate the potential clinical benefit of various therapeutic strategies. These strategies include those designed to increase deoxyhemoglobin S solubility; to increase erythrocyte volume or water content, thereby reducing the intracellular hemoglobin concentration; or, most recently, to decrease the proportion of hemoglobin S by increasing the amount of non-S hemoglobin. Increasing levels of hemoglobin F is of particular interest due to its "sparing" effect in inhibiting polymerization, the well-characterized epidemiological associations of high levels of hemoglobin F with reduced disease severity, and recent studies of drug-induced increases in hemoglobin F. Our analyses of equilibrium polymer formation at physiological oxygen saturation values suggest that small decreases in polymer formation at intermediate levels of hemoglobin F may give rise to a small decrease in anemia (as associated with homozygous alpha-thalassemia coexistent with sickle cell anemia), but that greater reductions in polymer formation may be necessary to effect a significant improvement in disease severity. Current studies of hydroxyurea-induced increases of hemoglobin F give cautious optimism that therapeutically useful levels may be attainable.

Anemia, Sickle Cell↗
PubMed1989

Hemodynamic studies in sickle cell disease.

With the availability of these noninvasive imaging modalities, one can now obtain more objective information about microcirculatory pathophysiology. As a result, previously held pathophysiologic paradigms in sickle cell disease will undoubtedly require modification or abandonment. These approaches should be viewed as supplements, not substitutes, for more traditional evaluations. Nonetheless, since these techniques are amenable to sequential application, these approaches promise to define disease severity more precisely, in quantitative terms, and should provide the means to follow the results of therapy unambiguously.

Anemia, Sickle Cell↗
PubMed1989

Cloning of cDNA from induced K562 cells which can activate globin gene expression.

A cDNA library from induced K562 cells was constructed and differentially screened for the isolation of clones encoding trans-acting factors which increase globin gene expression. The current study assumes that induced K562 cells contain transcriptionally active factors specific for globin genes which are absent or present only at very low levels in uninduced K562 cells. Upon screening the recombinant library, 75 cDNA clones hybridized specifically with cDNA probes from induced K562 cells and hybridized only slightly or not at all with cDNA probes from uninduced K562 cells and HL-60 cells, or from globin genes. Forty-five of the cDNA clones were full length complements to the corresponding RNA. To screen for trans-acting factors which can activate globin gene expression, the cDNA clones were inserted into a eukaryotic expression vector and co-transfected into HeLa cells with another vector containing the epsilon globin promoter 5' to the bacterial CAT (chloramphenicol acetyl transferase) reporter gene. Partial screening of the 45 full length clones revealed one cDNA (clone #17) that was able to increase CAT activity (driven by the epsilon-globin promoter) by about 2.5 times. This cDNA is 522 nucleotides in length and contains a long open reading frame. Examination with known DNA sequences indicates greater than 95% homology with the ferritin heavy chain.

Base Sequence↗
PubMed1989

In vitro transcription with K562 cell nuclear extract and globin genes.

Cloned human epsilon-, A/gamma- and beta-globin genes, the insulin gene and the adenovirus 2 major late promoter (Ad2MLP) were employed for transcription in vitro with K562 nuclear extracts. Nuclear extracts could direct accurate initiation of transcription from epsilon-globin without supplement of a whole cell extract. A clear dependence of protein concentration of nuclear extracts on transcriptional enhancement was observed with the epsilon-globin gene. To examine the cis-acting DNA sequences 5' of the promoter region which may be important in specific expression of globin genes, the epsilon-globin template was truncated using restriction enzymes. Transcriptional activity of the epsilon-globin gene varied according to the truncation suggesting possible regions to which nuclear proteins involved in transcription may bind. Fractionation of nuclear extracts by ion exchange chromatography indicated that activity could be recovered in a 175 mM ammonium sulfate fraction while the 50 mM ammonium sulfate fraction decreased transcription activity. A/gamma globin gene and Ad2MLP could be transcribed in nuclear extracts at higher concentrations, however, beta-globin and the insulin gene were not transcribed at any concentration assayed, either from induced or uninduced cells. Transcription of the beta-globin gene was observed in vitro when K562 nuclear extracts were supplemented with HeLa whole cell extracts.

Cell Nucleus↗
PubMed1988

In vitro differential expression of human globin genes.

K562 human erythroleukemia cells can be induced by hemin and other stimuli to produce hemoglobin and actively express epsilon- and gamma-globin genes but not the adult-like beta-globin gene. The control of globin gene expression is regulated by both cis-acting DNA sequences, such as the 5'-promoter region and 3'- and 5'-enhancers, and trans-acting factors. The activation of 5'-promoter DNA sequences mediated by trans-acting factors can be examined in a cell-free system by in vitro transcription. We report here the differential transcriptional activation of globin genes by nuclear extracts from K562 cells without the need of supplemental HeLa whole cell extracts. Transcription of the epsilon-globin gene which is more active in K562 cells than the gamma-globin gene was greater at low nuclear extract concentrations (protein concentration less than 1.5 mg/ml) than the gamma-globin gene. However, neither beta-globin nor insulin promoters (which are not active in intact K562 cells) were active in the nuclear extract in vitro transcription system. The accurate initiation and specific transcription of globin genes by K562 nuclear extracts suggest that trans-acting factors interacting with the 5'-promoter gene sequence regulate in part the extent and specificity of gene expression and that the activity of these factors can be preserved during their isolation.

Adenoviridae↗
PubMed1988

Use of selective vasodilation in treatment of sickle cell disease.

Individuals with sickle cell disease and its genetic variants demonstrate a modest to severe hemolytic anemia. They are also prone to both acute and chronic vaso-occlusive phenomena, which may lead to irreversible tissue damage, organ failure, and premature death. To better understand these vaso-occlusive phenomena, it would be useful to determine the extent to which arteriolar microvascular obstruction might contribute to the pathogenesis of sickle cell disease. We studied the hematologic and ocular effects of orally administered nifedipine in several steady-state patients and compared these with a case-control group of patients not receiving nifedipine. In the nifedipine-treated patients, a striking reversal in ischemic retinal and conjunctival changes was observed, as well as a significant improvement in color vision performance. In addition, the treated subjects showed a slight but significant decline in the levels of both indirect serum bilirubin and plasma hemoglobin, suggesting a concurrent amelioration in the rate of hemolysis. These observations are consistent with the hypothesis that inappropriate vaso-constriction, or frank vasospasm, perhaps in response to the altered rheology of red cells containing polymerized hemoglobin S, may play an important role in the pathophysiology of sickle cell disease. Furthermore, selective vasodilation may prove to be a useful adjunctive approach to the treatment of the manifestations of the sickle syndromes.

Adult↗
PubMed1988

Polymerization of sickle cell hemoglobin at arterial oxygen saturation impairs erythrocyte deformability.

We have examined the filterability of sickle erythrocytes, using an initial-flow-rate method, to determine whether sufficient hemoglobin S polymer forms at arterial oxygen saturation to adversely affect erythrocyte deformability. The amount of intracellular polymer was calculated as a function of oxygen saturation to estimate the polymerization tendency for each of eight patients with sickle cell anemia (SCA). Progressive reduction of oxygen tension within the arterial range caused a sudden loss of filterability of SCA erythrocytes through 5-micron-diam pores at a critical PO2 between 110 and 190 mmHg. This loss of filterability occurred at a higher PO2 than did morphological sickling, and the critical PO2 correlated significantly (r = 0.844-0.881, P less than 0.01) with the polymerization tendency for each patient. Study of density-gradient fractionated cells from four SCA patients indicated that the critical PO2 of dense cells was reached when only a small amount of polymer had formed, indicating the influence of this subpopulation on the results obtained for unfractionated cells. Impairment of erythrocyte filterability at high oxygen saturation (greater than 90%) suggests that small changes in oxygen saturation within the arterial circulation cause rheological impairment of sickle cells.

Anemia, Sickle Cell↗
PubMed1988

Is there a role for selective vasodilation in the management of sickle cell disease?

To test the hypothesis that microvascular obstruction to blood flow at the level of the arteriole may be significant in individuals with sickle cell anemia, the ophthalmologic effects of orally administered nifedipine were monitored in 11 steady-state patients. Three patients with evidence of acute peripheral retinal arteriolar occlusion displayed a prompt reperfusion of the involved segment. Two other patients showed fading of retroequatorial red retinal lesions. Color vision performance was improved in six of the nine patients tested. The majority of patients also demonstrated a significant decrease in the amount of blanching of the conjunctiva which reflects improved blood flow to this frequently involved area. Such improvements were not observable in a control group of untreated stable sickle cell subjects. These findings support the hypothesis that inappropriate vasoconstriction or frank vasospasm may be a significant factor in the pathogenesis of the microvascular lesions of sickle cell disease and, further, that selective microvascular entrapment inhibition may offer an additional strategy to the management of this disorder. We believe a larger, placebo-controlled study with nifedipine and similar agents is warranted.

Adult↗