Flow-cytometric enumeration of reticulocytes with the new fluorochrome 1′,3′-diethyl-4,2′-quinolylthiacyanine

Summary Several flow-cytometric methods for reticulocyte enumeration in whole blood have been developed, with different degrees of practical use. Recently, a new fluorochrome, 1,3-diethyl-4,2-quinolylthyacianine (DEQTC) was proposed in a brief report, as an alternative to thiazole orange for reticulocyte counting. We have evaluated the usefulness of this fluorescent stain by assessing the optimal conditions for the flow-cytometric analysis, and by comparing in double-blind assays the quantitative results of this technique with those obtained by manual counting with brilliant cresyl blue. Our results show that flow cytometry with DEQTC is highly correlated to the manual method (r=0.95–0.99), supporting the interest of this particular stain and of flow cytometry for routine laboratory work in hematology.     

A Study of δ-Globin Gene Mutations in the UK Population: Identification of Three Novel Variants and Development of a Novel DNA Test for Hb A′2

We report here the spectrum of δ-globin gene mutations found in the UK population. Nine different δ chain variants and two δ-thalassemia (δ-thal) mutations were characterized in a study of 127 alleles in patients with either a low Hb A₂ value or a split Hb A₂ peak on high performance liquid chromatography (HPLC). The most common δ chain variant was Hb A′₂ (or Hb B₂) [δ16(A13)Gly?→?Arg; HBD: c.49G?>?C] (77.0%), followed by Hb A₂-Yialousa [δ27(B9)Ala?→?Ser; HBD: c.82G?>?T] (12.0%), Hb A₂-Babinga [δ136(H14)Gly?→?Asp; HBD: c.410G?>?A] (3.0%), Hb A₂-Troodos [δ116(G18)Arg?→?Cys; HBD: c.349C?>?T] (1.0%), Hb A₂-Coburg [δ116(G18)Arg?→?His; HBD: c.350G?>?A] (2.0%) and Hb A₂-Indonesia [δ69(E13)Gly?→?Arg; HBD: c.208G?>?C] (1.0%). Three novel variants were identified: Hb A₂-Calderdale [codon 2 (CAT?>?AAT), His?→?Asn; HBD: c.7C?>?A], Hb A₂-Walsgrave [codon 52 (GAT?>?CAT), Asp?→?His; HBD: c.157G?>?C] and Hb A₂-St. George’s [codon 81 (CTC?>?TTC), Leu?→?Phe; HBD: c.244C?>?T]. In addition, two known δ-thal mutations were observed: ?68 (C?>?T); HBD: c.-118C?>?T and codon 4 (ACT?>?ATT); HBD: c.14C?>?T. Amplification refractory mutation system (ARMS) primers were developed to provide a simple molecular diagnostic test for the most common variant, Hb A′₂. Three of the variants had a characteristic HPLC retention time that can be used for a presumptive diagnosis.     

2′,3′-双去氧胞苷导致中毒性神经病

作者报告了52例2',3'-双去氧咆苷(ddc)所致中毒性神经病的临床表现和试验结果。方法 52例 AIDS 患者接受下述4种剂量的 ddc 治疗:①ddc0.06mg/kg/4h(14例);②     

Differentiation of Dunn osteosarcoma cells in response to dibutyryl cyclic 3′,5′-adenosine monophosphate

We investigated the effects of dibutyryl cyclic adenosine 3,5-monophosphate (Bt₂cAMP) on the differentiation of Dunn osteosarcoma cells. Flow-cytometric analysis and DNA synthesis assay showed that Bt₂cAMP decreased the cell population in the S phase in a time- and dose-dependent manner. Also, the cells showed distinct morphological and functional alterations: the cell morphology changed to a fibroblast-like appearance with long and thin protoplasmic processes, the knobs or blebs on both the cell membrane and nuclear membrane disappeared and the intracellular alkaline phosphatase activity increased. Moreover, Bt₂cAMP-treated cells secreted a large quantity of fibronectin, which was deposited on the extended cell surface in the culture medium. Thus, Dunn osteosarcoma cells are differentiated morphologically and functionally by Bt₂cAMP, and might be transformed to benign precursor cells.Abbreviation Bt₂cAMP dibutyryl cyclic 3,5-adenosine monophosphate This research was supported by a grant-in-aid for eucouragement of young scientists from the Japanese Ministry of Education, Science and Culture (02770900)     

Association of homozygous SDF-1 3′A genotype with proliferative diabetic retinopathy

Stromal cell-derived factor 1 (SDF-1) is involved in the development of experimental proliferative retinopathy. Since little data are available on the genetic predisposition or on biomarkers predicting the development of proliferative retinopathy, we assessed the distribution of the SDF-1 3′A genotype in 130 diabetic patients with retinopathy. In patients with proliferative retinopathy, the frequency of the homozygous SDF-1 3′A genotype was significantly higher than in patients with non-proliferative retinopathy (10.9% of PDR vs. 0 of NPDR, P = 0.01). This association was confirmed when type 2 diabetes patients were analysed separately (10.3% of PDR vs. 0 of NPDR, P = 0.03). The finding that homozygous carriers of the SDF-1 3′A genotype are more frequent in diabetes patients with proliferative retinopathy suggests a possible role of this genotype in the development of sight-threatening diabetic retinopathy.     

Indomethacin increases 2′,5′-oligoadenylate synthetase release by cultured liver tissue of patients with HCV chronic active hepatitis

Interferon-α (IFN-α) stimulates the synthesis of antiviral proteins, such as 2′,5′-oligoadenylate synthetase (2′5′-OAS), and activation of arachidonic acid (AA) metabolism seems to constitute the post-receptorial signal induced by this cytokine. We evaluated the in vitro effect of IFN-α, indomethacin (an inhibitor of cyclooxygenase pathway of AA) and IFN-α plus indomethacin on 2′5′-OAS release by cultured liver biopsies of patients with active or inactive HCV infection and uninfected patients with various liver diseases. The results demonstrated that basal 2′5′-OAS release was significantly higher in patients with active HCV infection with respect to uninfected ones. In two HCV-Ab positive but HCV-RNA negative patients, basal 2′5′-OAS was similar to uninfected patients. In HCV-RNA positive patients, indomethacin alone was able to significantly increase the 2′5′-OAS production in a similar fashion to IFN-α alone, while a synergistic effect on 2′5′-OAS increase was seen when biopsies were cultured with IFN-α plus indomethacin. A similar trend was observed in the uninfected and in the HCV-Ab positive but HCV-RNA negative patients, even if the increase did not reach statistical significance. These results confirm the interaction between IFN-α and AA metabolites and suggest an evaluation of the in vivo response of IFN-α plus nonsteroid antiinflammatory drugs combined therapy.     

Identification of a Novel Mutation in the β-Globin Gene 3′ Untranslated Region (HBB: c.*+118A > G) in Spain

Abstract

The 3′ untranslated region (3′UTR) region is well known to be associated with mRNA stability because of its associations with 3′ end processing, polyadenylation and mRNA capping. Mutations located in this area cause a β-thalassemia (β-thal) phenotype compatible with β⁺-thal. Two brothers, 49- and 41-years-old, were diagnosed with β-thal intermedia (β-TI) at 2 years of age. The β-globin gene from the promoter region to the 3′UTR was sequenced and both brothers were diagnosed to be compound heterozygotes for the 3′UTR +1592 (A?>?G) (HBB: c.*+118A?>?G) and codon 39 (C?>?T) (HBB: c.118C?>?T) mutations. Their mother was a carrier of the nonsense codon 39 mutation and her hematological data suggested β-thal trait; their father was a carrier of the 3′UTR +1592 mutation, though he did not have hematological parameters associated with β-thal. The adenine at position +1592 or +118 bases downstream of the termination codon is highly conserved among primates and placental mammals, as it is located between the polyadenylation A signal (PAS) and the polyadenylation A cleavage (PAC) sites. Given its location, it is likely that this mutation would interfere with mRNA maturation; however, the clinical data of the heterozygous carriers show virtually no significant alterations. Therefore, we suggest that the impact on cleavage-stimulation factor (CstF) recognition of the mRNA sequence would be minimal and not significantly alter polyadenylation. Although the mechanism is not known, and because the carrier has no β-thal minor, the mRNA is stable enough that the synthesis of the β-globin chain is unaffected.     

Inactivity of human β,β-carotene-9′,10′-dioxygenase (BCO2) underlies retinal accumulation of the human macular carotenoid pigment

The macula of the primate retina uniquely concentrates high amounts of the xanthophyll carotenoids lutein, zeaxanthin, and meso-zeaxanthin, but the underlying biochemical mechanisms for this spatial- and species-specific localization have not been fully elucidated. For example, despite abundant retinal levels in mice and primates of a binding protein for zeaxanthin and meso-zeaxanthin, the pi isoform of glutathione S-transferase (GSTP1), only human and monkey retinas naturally contain detectable levels of these carotenoids. We therefore investigated whether or not differences in expression, localization, and activity between mouse and primate carotenoid metabolic enzymes could account for this species-specific difference in retinal accumulation. We focused on β,β-carotene-9′,10′-dioxygenase (BCO2, also known as BCDO2), the only known mammalian xanthophyll cleavage enzyme. RT-PCR, Western blot analysis, and immunohistochemistry (IHC) confirmed that BCO2 is expressed in both mouse and primate retinas. Cotransfection of expression plasmids of human or mouse BCO2 into Escherichia coli strains engineered to produce zeaxanthin demonstrated that only mouse BCO2 is an active zeaxanthin cleavage enzyme. Surface plasmon resonance (SPR) binding studies showed that the binding affinities between human BCO2 and lutein, zeaxanthin, and meso-zeaxanthin are 10- to 40-fold weaker than those for mouse BCO2, implying that ineffective capture of carotenoids by human BCO2 prevents cleavage of xanthophyll carotenoids. Moreover, BCO2 knockout mice, unlike WT mice, accumulate zeaxanthin in their retinas. Our results provide a novel explanation for how primates uniquely concentrate xanthophyll carotenoids at high levels in retinal tissue.The macular pigment of the human retina consists of the xanthophyll carotenoids, lutein, zeaxanthin, and their metabolite, meso-zeaxanthin (1–3). Macular pigments are highly concentrated in the foveal area of the human retina. Their highest concentration can reach 1 mM, making the macula a visible yellow spot at the center of the human retina. These xanthophylls are known to play antioxidant and light-screening roles in nature (4–6). Clinical studies have shown that lutein and zeaxanthin supplementation can reduce the risk of age-related macular degeneration (AMD), a leading cause of blindness in the developed world (4, 7, 8). Recently, the Age-Related Eye Disease Study 2 (AREDS2) showed that daily oral supplementation with 10 mg lutein and 2 mg zeaxanthin was associated with 10–18% reduced progression to advanced AMD over the 5-y study relative to original Age-Related Eye Disease Study (AREDS) formulations without lutein and zeaxanthin, and the authors recommended adding lutein and zeaxanthin to replace β-carotene in the formulation for safety and efficacy reasons (9, 10).The spatially and chemically specific accumulation of high concentrations of xanthophylls in the retina is a unique feature of the primate retina relative to other mammals, but the biochemical basis for this high degree of specificity is unclear. High-affinity binding proteins might drive this process. Mice and humans express a zeaxanthin-binding protein, the pi isoform of glutathione S-transferase (GSTP1), abundantly in the retina, yet only the human retina naturally contains zeaxanthin and meso-zeaxanthin (11, 12). Thus, other biochemical mechanisms underlying accumulation of the human macular pigment need to be considered.β,β-Carotene-9′,10′-dioxygenase (BCO2, also known as BCDO2) is a xanthophyll carotenoid cleavage enzyme which can cleave carotenoids in many animals eccentrically at the 9–10 or 9′-10′ carbon–carbon double bonds into C13- and C27-apocarotenoids, or further into C14-dialdehyde (13, 14). Its physiological function is supported by genetic evidence from several species. A nonsense mutation in the sheep BCO2 gene that introduces a stop codon at amino acid 66 of the 575-aa full-length protein is strongly associated with a yellow fat phenotype due to the high amount of lutein that deposits in tissue (15). Cis-acting and tissue-specific regulatory mutations that inhibit the expression of BCO2 in domestic chickens can cause them to have yellow skin secondary to carotenoid accumulation (16). Genetic variations in the BCO2 gene have been reported to increase carotenoid levels in bovine adipose tissue and milk (17). In vitro biochemical studies have also demonstrated that ferret BCO2 can cleave lutein and zeaxanthin (13).BCO2, β,β-carotene-15,15′-monooxygenase (BCO1), and retinal pigment epithelium-specific 65 kDa protein (RPE65) are members of the polyene oxygenase gene family. BCO1, which is also known as BCMO1, cleaves β-carotene symmetrically at the 15–15′ position to yield two molecules of retinal, the key pigment in the visual cycle, and it is inactive with xanthophyll (oxygenated) carotenoids such as lutein and zeaxanthin (18, 19). RPE65, an essential enzyme in the visual cycle, participates in the conversion of all-trans-retinyl palmitate to 11-cis-retinol, and various mutations are associated with Leber congenital amaurosis type 2 and retinitis pigmentosa (18, 20–22). BCO2 may also play important roles in ocular tissues, but a physiological function has not been established. Based on the fact that high amounts of lutein and zeaxanthin exist in the human macula, we hypothesized that either human BCO2 is not expressed in human retina or it is an inactive carotenoid cleavage enzyme. In this study, we tested these proposed hypotheses by investigating the expression, distribution, and activity of BCO2 in human, monkey, and mouse retina.     

A new subtype of 3′ region of cagA gene in Helicobacter pyloristrains isolated from Zhejiang Province in China

AIM: To isolate the subtypes of 3′ region of cagA gene in Helicobacter pylori (H pylon) strains from Zhejiang Province in China and to investigate their relations to H pylori-associated gastroduodenal diseases. METHODS: One hundred and thirty-seven H pylori clinical strains were isolated from the gastric mucosa specimens of 74 patients with chronic gastritis, 61 with peptic ulceration, and 2 with gastric cancer. Bacterial genomic DNA was extracted and 3′ region of cagA gene was amplified by polymerase chain reaction (PCR). Subtypes of 3′ region of cagA gene were determined by the size of PCR amplified segments. The sequences of the subtypes were analyzed by PCR-based sequencing. RESULTS: Of the 137 H pyloriisolates from Zhejiang Province, 132 (96.4%) yielded PCR products that could be classified into three groups of subtypes, named as subtypes Ⅰ, Ⅱ, and Ⅲ according to their sizes. The sizes of subtypes Ⅰ, Ⅱ, and Ⅲ were 648-650bp, 705-707bp, and 815bp, respectively. Among the 132 cagA-positive H pylori strains, 123(93.2%) belonged to the group of subtype Ⅰ, 6 (4.5%) presented subtype Ⅱ, 1(0.8%) was subtype Ⅲ, and 2(1.5%) presented subtypes Ⅰ and Ⅲ both. The primary structure of subtype Ⅰ was composed of 3 repeats of R1, 1 repeat of R2 and 1 repeat of R3. Subtype Ⅱ possessing 4 repeats of R1, 2 repeats of R2 and 1 repeat of R3 was a newly found type of 3′ region of cagA gene which had not been reported before. The primary structure of subtype Ⅲ consisted of 4 repeats of R1, Ⅰ repeat of R2 and 2 repeats of R3. Comparison of the sequences of subtype Ⅰ strains with the corresponding sequences deposited in GenBank, showed a similarity of 95.0% (94.0-96.1%) for nucleotide sequences and 95.9% (94.9-97.4%) for deduced amino acid sequences. Comparison of the sequences of subtype Ⅲ strains with the corresponding sequences deposited in GenBank, showed a similarity of 93.9% (90.8-96.9%) for nucleotide sequences and 93.2% (90.2-96.2%) for deduced amino acid sequences. Among subtype Ⅱ strains, the nucleotide and deduced amino acid sequences showed a similarity of 95.2% (94.1-96.5%) and 96.4% (93.8-97.9%),respectively. There were no statistical differences in the distribution of subtypes of 3′ region of cagA gene among different H pylori-associated gastroduodenal diseases (x^2=11.544, P>0.05). CONCLUSION: There are three subtypes (Ⅰ,Ⅱ, and Ⅲ) of 3′ region of cagA gene in Hpylori strains isolated from Zhejiang Province, and subtype Ⅰ is predominant. Subtype Ⅱ is a newly found subtype of 3′ region of cagA gene. The result of this study does not support the view that the subtypes of 3′ region of cagA gene in H pylori isolated from Zhejiang Province are correlated with the clinical outcomes of H pylori infection.     

Hematological and Molecular Characterization of a Novel Hb A2 Variant with Homozygous α-Thalassemia-2 in a Southern Thai Individual

We report here the hematological and molecular features of a novel δ-globin chain variant found in a Southern Thai woman. Her complete blood count was as follows: red blood cell (RBC) count 5.90?×?10¹²/L, hemoglobin concentration (Hb) 12.6?g/dL, packed cell volume (PCV) 0.41?L/L, mean corpuscular volume (MCV) 69.5 fL, mean corpuscular Hb (MCH) 21.4?pg, mean corpuscular Hb concentration (MCHC) 30.7?g/dL and RBC distribution width (RDW) 13.1%. The blood smear demonstrated microcytic hypochromic RBCs suggestive of thalassemia trait. Hemoglobin analysis identified Hb A₂?+?Hb A₂-Kiriwong (2.4%) and Hb F (0.1%) on high performance liquid chromatography (HPLC). To characterize the α-thalassemia (α-thal) genotype, common α-thal-1 and α-thal-2 alleles were characterized by multiplex gap-polymerase chain reaction (gap-PCR). The results revealed homozygous α-thal-2 (–α^3.7/–α^3.7) in this case. DNA sequencing showed the presence of a novel δ-globin gene mutation [δ77(EF1)His→Arg; HBD: c.233A>G] that we named Hb A₂-Kiriwong for the village from where the proband lived. In summary, the presence of microcytic hypochromic RBCs in this case was likely the result of the homozygous –α^3.7 (rightward) deletion and was not affected by this Hb A₂ variant.     

Hb A2 Hong Kong – A Novel δ-Globin Variant in a Chinese Family Masks the Diagnosis of β-Thalassemia Trait

A 42-year-old Chinese woman (FP) was the mother of a patient with β-thalassemia major (β-TM) due to a compound heterozygosity for β⁰-thalassemia (β⁰-thal) mutations. She was also found to have a low Hb A₂ level of 1.6% by high performance liquid chromatography (HPLC) despite being a heterozygous carrier of the codons 41/42 (–TCTT) (HBB:c.126_129delCTTT) β⁰-thal mutation. Doubling the amount of hemolysate loaded for chromatography revealed a widened Hb A₂ peak and raised the level to 4.1%, consistent with β-thal trait. Direct nucleotide sequencing detected a novel δ-globin gene mutation at codon 29 (HBD:c.89G>A), which leads to a glycine to aspartic acid substitution. A homologous mutation at codon 29 in the β-globin gene [Hb Lufkin or β29(B11)Gly→Asp] has been reported in Black families. This report highlights the importance of genotype-phenotype correlation and the potential pitfall of relying on Hb A₂ level for phenotypic diagnosis of β⁰-thal trait.     

Effect of 5- AZn-2 ′-deoxycytidine on proliferation of human lung adenocarcinoma cell line A549 in vitro

ObjectiveTo explore effect of 5-AZn-2 ′-deoxycytidine on proliferation of human lung adenocarcinoma cell line A549 in vitro.MethodsSuperoxide dismutase (SOD) activity was measured by hydroxylamine colorimetric method. Inhibition effect of 5-AZn-2′ deoxycytidylic acid at different concentration and different time on growth of A549 cell was determined by MTT assay. Methylene dioxyamphetamine (MDA) was measured by thiobarbituric acid colorimetric method. Effect of 5-AZn-2′ deoxycytidylic acid on apoptosis of A549 cell was determined by Hoechst 33258 dyeing detection.Results5-AZn-2′ deoxycytidylic acid had significant inhibition effect on proliferation of A549 cells in vitro, and the inhibition was notably dependent on time and dosage during 48-72 h; SOD level was significantly lower than those of control group (P<0.05, P<0.01), MDA level was significantly higher than those in the control group (P<0.05, P<0.01). A549 cells began to be in apoptosis after using 5-AZn-2′deoxycytidylic acid.Conclusions5- AZn-2′ deoxycytidylic acid has significant inhibition effect on growth of A549 cell, and can lead the change of lipid peroxidation. It indicates that the mechanism has relationship with A549 cell cycle tissue and induction factor of apoptosis.     

Hemoglobin A2′ (HbA2δ′) in the Mauritanian population: first results of a preliminary survey

The Mauritanian population consists of two main groups: one Arab and Berber population and one of Black African origin. The latter includes five ethnic groups: Pular, Soninke, Black Maurs, Wolof, and Bambara. Abnormal HbA2 was found in the Mauritanian population and was characterized by cellulose acetate electrophoresis at pH 8.4 followed by DNA analysis. Among 785 subjects examined, 17 were carriers of HbA2' corresponding to 2.16 percent. The highest frequency was observed among the Pular (3.09 percent), the Black Maurs (2.72 percent), the Wolof (2.27 percent), and the Soninke (2.04 percent).     

Origin of Hb A2′ (Hb B2) [δ16(A13)Gly→Arg (GGC→CGC)]

On a field trip to the Dogon country (le Pays Dogon) in central Mali, we detected a high frequency of the Hb A₂ abnormality, reaching higher numbers among blacksmiths (up to 12.4%) living in the same villages. In this report, by direct nucleotide sequencing and employing a polymerase chain reaction‐restriction fragment length polymorphism approach, we show that the Hb A₂ variant observed in the Dogon population is indeed Hb A₂^′, also called Hb B₂, and that in all of the cases the abnormal δ‐globin gene is linked to a unique haplotype. The same haplotype was found linked to Hb A₂^′ in the Herero population belonging to the South African Bantu‐speaking Blacks from Namibia. Although the unique origin of this mutation in Africa is a possibility, a recurrent mutational event cannot be excluded because the linked β cluster haplotype is one of the two major haplotypes found in all African populations. A study of populations from other regions of Africa is required to clarify this issue.     

A GG allele of 3′-side AKT1 SNP is associated with decreased AKT1 activation and better prognosis of gastric cancer

Purpose

v-akt Murine thymoma viral oncogene homolog (AKT) pathway is critically involved in cancer cell growth, invasion, and survival. We examined the correlation between the genetic variations in molecules of AKT pathway and clinical outcomes of gastric cancer.

Patients and methods

Six single nucleotide polymorphisms (SNPs) located in the four core genes of AKT pathway, namely the PIK3CA, PTEN, AKT1, and mTOR, were determined in 221 patients with stage T2 and T3 gastric cancer. Additionally, the activation of AKT1 in gastric cancer tissues was examined by immunostaining. The correlation between SNPs, AKT activation, and the progress of gastric cancer was analyzed after an average of 51-month follow-up.

Results

The overall recurrence and survival rate in this study group were 54.8 and 46.6 %, respectively. The recurrence rate was reduced 30.4 %, and the survival rate was increased 33.7 % in patients with GG allele of a 3′-side AKT1 SNP (rs2498804). Significantly, GG allele was associated with lower AKT1 activation in gastric cancer tissues. On the contrary, CC allele of PTEN (rs701848) was associated with the increased risk of recurrence (hazard ratio [HR] 2.06, 95 % CI 1.19–3.58) and patient death (HR 2.01, 95 % CI 1.15–3.53).

Conclusions

The genetic variants in the PI3K/PTEN/AKT especially the GG allele in 3′ side of AKT1 are closely related to clinical outcomes of gastric cancer.     

MCI-154: A Second Generation Ca2+ Sensitizer That Does Not Impair Relaxation — A Novel Approach to the Treatment of Heart Failure

With the negative results of the cyclic AMP-dependent positive inotropic agents in clinical trials, great interest has been focused on the development of agents that directly activate cardiac myofilaments. These drugs are called “Ca²⁺ sensitizers”; they are expected to represent a possible new pharmacological approach to the therapy of chronic heart failure. MCI-154 is one of the most powerful and promising Ca²⁺ sensitizers currently in clinical trials. In preclinical studies, the positive inotropic action of MCI-154 was observed at the concentrations at which the drug did not increase intracellular Ca²⁺. In skinned cardiac muscle fiber bundles from various animal species MCI-154 shifted p_Ca(-log [Ca²⁺] M)-force relation upward and to the left, suggesting that this drug not only increases sensitivity of myofibrils to Ca²⁺, but also enhances cross-bridge interaction. In regard to the molecular mechanism of action of MCI-154, the earliest experiments showed that MCI-154 at the high concentration (10⁻⁴ M) stimulated binding of Ca²⁺ to troponin (Tn) C. However, MCI-154-(at concentrations lower than 10⁻⁵ M)-induced increase in the affinity of TnC to Ca²⁺ led to the complex formation of TnC with TnI and TnT. This effect of the drug on Ca²⁺ regulation and interaction between troponin subunits was discovered using fluorescence spectroscopy. At 10⁻⁴ M MCI-154 decreased binding of Ca²⁺ to TnC. The Ca²⁺ sensitizing effect of MCI-154 disappeared when cardiac TnI was exchanged for skeletal TnI. Taken together, it can be concluded that TnI may represent a target protein for the positive inotropic action of MCI-154. The long-term treatment with MCI-154 prolonged the life span of cardiomyopathic hamsters, Biol 14.6, a model that resembles human heart failure. Unlike PDE inhibitors, MCI-154 did not aggravate arrhythmias generated in the two-stage coronary ligation-, digitalis- and catecholamine-induced canine arrhythmia models. In clinical studies MCI-154 improved contractile function in patients with left ventricular dysfunction after myocardial infarction. Its effect was not associated with an increase in myocardial oxygen consumption or impaired relaxation. In conclusion, MCI-154 could be promising in the treatment of chronic heart failure. Clinical studies with MCI-154 are currently in progress.