Characteristics of Nonunion After Arthroscopic Ankle Arthrodesis: A Multicenter Retrospective Cohort Study.

Nonunion is a major complication of arthroscopic ankle arthrodesis. However, the characteristics and risk factors of nonunion are not well understood. This retrospective multicenter observational study aimed to clarify the characteristics of nonunion after arthroscopic ankle arthrodesis. We included 154 patients who underwent arthroscopic ankle arthrodesis at any 1 of 5 institutions. Patients were divided into 2 groups: union and nonunion, and the groups were compared. Age, sex, body mass index, diabetes, smoking, corticosteroid use, diagnosis, treatment information, treatment protocol, radiographic evaluation, and patient-reported outcomes were recorded and analyzed. On radiographs, bony union was observed in 142 ankles (91.0%) but not in 12 ankles (9.0%). Postoperative radiographic tibial bony gap (mm) was significantly larger in the nonunion group (medial = 1.98, center = 1.65, anterior = 2.21, middle = 1.72, posterior = 3.01) than in the union group (medial = 1.35, center = 1.13, anterior = 1.28, middle = 1.03, posterior = 2.03). Furthermore, the visual analog score (VAS) of pain and pain-related self-administered foot evaluation questionnaire (SAFE-Q) subscale score significantly worsened in the nonunion group (VAS = 3.83, SAFE-Q subscale score = 69.8) compared to that in the union group (VAS = 1.35, SAFE-Q subscale score = 76.6). A larger radiographic tibiotalar bony gap was observed in the nonunion group. Other measurement outcomes were not associated with nonunion. Additionally, patient-reported outcomes markedly worsened in the nonunion group.

EZH1/2 dual inhibitors suppress HTLV-1-infected cell proliferation and hyperimmune response in HTLV-1-associated myelopathy.

Background: Human T-cell leukemia virus type 1 (HTLV-1) causes HTLV-1-associated myelopathy (HAM), adult T-cell leukemia/lymphoma (ATL), HTLV-1-associated uveitis, and pulmonary diseases. Although both HAM and ATL show proliferation of infected cells, their pathogeneses are quite different. In particular, the pathogenesis of HAM is characterized by hyperimmune responses to HTLV-1-infected cells. Recently, we demonstrated the overexpression of histone methyltransferase EZH2 in ATL cells and the cytotoxic effects of EZH2 inhibitors and EZH1/2 dual inhibitors on these cells. However, these phenomena have never been studied in HAM. Furthermore, what effect these agents have on the hyperimmune response seen in HAM is completely unknown. Methods: In this study, we investigated histone methyltransferase expression levels in infected cell populations (CD4+ and CD4+CCR4+ cells) from patients with HAM using microarray and RT-qPCR analyses. Next, using an assay system that utilizes the spontaneous proliferation characteristic of peripheral blood mononuclear cells derived from patients with HAM (HAM-PBMCs), we investigated the effects of EZH2 selective inhibitors (GSK126 and tazemetostat) and EZH1/2 dual inhibitors (OR-S1 and valemetostat, also known as DS-3201), particularly on cell proliferation rate, cytokine production, and HTLV-1 proviral load. We also examined the effect of EZH1/2 inhibitors on the proliferation of HTLV-1-infected cell lines (HCT-4 and HCT-5) derived from patients with HAM. Results: We found elevated expression of EZH2 in CD4+ and CD4+CCR4+ cells from patients with HAM. EZH2 selective inhibitors and EZH1/2 inhibitors significantly inhibited spontaneous proliferation of HAM-PBMC in a concentration-dependent manner. The effect was greater with EZH1/2 inhibitors. EZH1/2 inhibitors also reduced the frequencies of Ki67+ CD4+ T cells and Ki67+ CD8+ T cells. Furthermore, they reduced HTLV-1 proviral loads and increased IL-10 levels in culture supernatants but did not alter IFN-γ and TNF-α levels. These agents also caused a concentration-dependent inhibition of the proliferation of HTLV-1-infected cell lines derived from patients with HAM and increased annexin-V(+)7-aminoactinomycin D(-) early apoptotic cells. Conclusion: This study showed that EZH1/2 inhibitors suppress HTLV-1-infected cell proliferation through apoptosis and the hyperimmune response in HAM. This indicates that EZH1/2 inhibitors may be effective in treating HAM.

Potential role of HTLV-1 Tax-specific cytotoxic t lymphocytes expressing a unique t-cell receptor to promote inflammation of the central nervous system in myelopathy associated with HTLV-1.

Human T-lymphotropic virus 1 (HTLV-1) infection causes two serious diseases: adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy (HAM). Immunological studies have revealed that HTLV-1 Tax-specific CD8+ cytotoxic T-cells (Tax-CTLs) in asymptomatic carriers (ACs) and ATL patients play an important role in the elimination of HTLV-1-infected host cells, whereas Tax-CTLs in HAM patients trigger an excessive immune response against HTLV-1-infected host cells infiltrating the central nervous system (CNS), leading to local inflammation. Our previous evaluation of HTLV-1 Tax301-309 (SFHSLHLLF)-specific Tax-CTLs (Tax301-309-CTLs) revealed that a unique T-cell receptor (TCR) containing amino acid (AA)-sequence motif PDR, was shared among HLA-A*24:02+ ACs and ATL patients and behaved as an eliminator by strong activity against HTLV-1. However, it remains unclear whether PDR+Tax301-309-CTLs also exist in HLA-A*24:02+ HAM patients and are involved in the pathogenesis of HAM. In the present study, by high-throughput TCR repertoire analysis technology, we revealed TCR repertoires of Tax301-309-CTLs in peripheral blood (PB) of HLA-A*24:02+ HAM patients were skewed, and a unique TCR-motif PDR was conserved in HAM patients (10 of 11 cases). The remaining case dominantly expressed (-DR, P-R, and PD-), which differed by one AA from PDR. Overall, TCRs with unique AA-sequence motifs PDR, or (-DR, P-R, and PD-) accounted for a total of 0.3-98.1% of Tax301-309-CTLs repertoires of HLA-A*24:02+ HAM patients. Moreover, TCR repertoire analysis of T-cells in the cerebrospinal fluid (CSF) from four HAM patients demonstrated the possibility that PDR+Tax301-309-CTLs and (-DR, P-R, and PD-)+Tax301-309-CTLs efficiently migrated and accumulated in the CSF of HAM patients fostering increased inflammation, although we observed no clear significant correlation between the frequencies of them in PB and the levels of CSF neopterin, a known disease activity biomarker of HAM. Furthermore, to better understand the potential function of PDR+Tax301-309-CTLs, we performed immune profiling by single-cell RNA-sequencing of Tax301-309-CTLs, and the result showed that PDR+Tax301-309-CTLs up-regulated the gene expression of natural killer cell marker KLRB1 (CD161), which may be associated with T-cell activation and highly cytotoxic potential of memory T-cells. These findings indicated that unique and shared PDR+Tax301-309-CTLs have a potential role in promoting local inflammation within the CNS of HAM patients.

Effect of Accelerated Rehabilitation on Early Return to Sport After Arthroscopic Ankle Lateral Ligament Repair.

Background: Although the minimal invasiveness of arthroscopic ankle lateral ligament repair (ALLR) means that an early return to sporting activities can be anticipated, studies have described postoperative cast immobilization and the avoidance of weightbearing for a certain period. Accelerated rehabilitation may be helpful for an early return to sport. Purpose: To investigate clinical outcomes of ALLR and accelerated rehabilitation with a minimum duration of postoperative ankle immobilization and proactive early weightbearing. Study Design: Case series; Level of evidence, 4. Methods: This study investigated 23 ankles of 22 patients (11 men, 11 women; mean age, 38.7 years) who underwent ALLR for chronic lateral ankle instability. Postoperative management included the avoidance of weightbearing until postoperative day 3, after which full weightbearing walking with a brace was permitted. The objective was to return to competitive sport 8 weeks after surgery. The following were evaluated: pre- and postoperative instability and pain symptoms, ankle range of motion, anterior drawer distance on stress radiograph, anterior translation measured with a capacitance-type strain sensor, the Ankle-Hindfoot Scale from the Japanese Society for Surgery of the Foot, and the SAFE-Q (Self-Administered Foot Evaluation Questionnaire). Results: Two male patients dropped out and were excluded from analysis. Postoperatively, instability and pain resolved or improved in all patients. There was no significant postoperative change in range of motion. There were significant pre- to postoperative improvements in talar tilt angle (from 12.2°-5.6°, P < .01), anterior drawer distance (8.2-4.4 mm, P < .01), and anterior translation (10.5-4.6 mm, P < .01) as well as the Ankle-Hindfoot Scale score (68.8-96.8, P < .01) and all subscales of the SAFE-Q (P ≤ .01 for all). Complete return to sport was achieved by 75% of the patients at 8 weeks postoperatively. Conclusion: When accelerated rehabilitation with proactive weightbearing exercises was implemented from postoperative day 3 without ankle immobilization after ALLR, there were significant improvements in objective assessments of ankle stability and clinical scores, and as many as 75% of the patients were able to make a complete return to sport within 8 weeks.

Efficacy of Corticosteroid Therapy for HTLV-1-Associated Myelopathy: A Randomized Controlled Trial (HAMLET-P).

Corticosteroids are most commonly used to treat HTLV-1-associated myelopathy (HAM); however, their clinical efficacy has not been tested in randomized clinical trials. This randomized controlled trial included 8 and 30 HAM patients with rapidly and slowly progressing walking disabilities, respectively. Rapid progressors were assigned (1:1) to receive or not receive a 3-day course of intravenous methylprednisolone in addition to oral prednisolone therapy. Meanwhile, slow progressors were assigned (1:1) to receive oral prednisolone or placebo. The primary outcomes were a composite of ≥1-grade improvement in the Osame Motor Disability Score or ≥30% improvement in the 10 m walking time (10 mWT) at week 2 for rapid progressors and changes from baseline in 10 mWT at week 24 for slow progressors. In the rapid progressor trial, all four patients with but only one of four without intravenous methylprednisolone achieved the primary outcome (p = 0.14). In the slow progressor trial, the median changes in 10 mWT were -13.8% (95% CI: -20.1--7.1; p < 0.001) and -6.0% (95% CI: -12.8-1.3; p = 0.10) with prednisolone and placebo, respectively (p for between-group difference = 0.12). Whereas statistical significance was not reached for the primary endpoints, the overall data indicated the benefit of corticosteroid therapy. (Registration number: UMIN000023798, UMIN000024085).

Surgically treated reactive arthritis of the ankle after COVID-19 infection: A case report.

A 37-year-old man developed right ankle pain and swelling six days after being diagnosed with coronavirus disease (COVID-19). Despite conservative treatment, his ankle symptoms persisted. Magnetic resonance imaging and computed tomography showed synovial hypertrophy and bone erosion in the ankle. Following arthroscopic synovectomy, performed 69 days after the COVID-19 diagnosis, the pain improved significantly. The clinical course was consistent with that of reactive arthritis following severe acute respiratory syndrome coronavirus 2 infection. The pathological findings resembled rheumatoid nodules. The bone erosion may have originated from the inflammatory pathway, which resembles the mechanism of rheumatoid arthritis.

Relationship Between Plantar Callosity and Foot Deformity in Hallux Valgus Using Weightbearing Computed Tomography.

Plantar callosities under lesser metatarsals are often accompanied by the hallux valgus, and the cause of callosity is thought to be associated with the foot deformity, such as the metatarsal length discrepancy, the abnormal metatarsal head height, cavus, flat foot, and rheumatoid conditions. However, it is unclear which variable is most involved in the cause of callosity in hallux valgus deformity. To clarify the factors associated with the callosity with hallux valgus deformity, we conducted multiple image assessments based on weightbearing radiography and computed tomography. A retrospective review was performed based on the collection of clinical records from all patients with hallux valgus treated from 2010 to 2019 in our institution. We measured the hallux valgus angle, intermetatarsal angles, calcaneal pitch angles, talo-first metatarsal angles, metatarsal length, metatarsal head height, first metatarsal pronation angles, and sesamoid position with weightbearing radiography and computed tomography. We analyzed the relation between callosity formation and imaging assessments using univariate and multivariate logistic regression models. Fifty feet were retrospectively evaluated, and multiple logistic analyses by the stepwise method revealed that the first metatarsal-lateral-sesamoid distance was the only radiographical variable associated with callosity formation among all the tested variables (p < .001). As the grade of the callosity became more severe, the lateral shift of the lateral sesamoid increased. The position of the sesamoid bone appears to have a critical role in the assessment and choice of treatment protocols and further research needs to be conducted on the relationship with the position of sesamoid bone to elucidate the mechanism of callus formation.

Two New Terpenes Isolated from Dictyostelium Cellular Slime Molds.

We report a protoilludane-type sesquiterpene, mucoroidiol, and a geranylated bicyclogermacranol, firmibasiol, isolated from Dictyostelium cellular slime molds. The methanol extracts of the fruiting bodies of cellular slime molds were separated by chromatographic methods to give these compounds. Their structures have been established by several spectral means. Mucoroidiol and firmibasiol are the first examples of more modified and oxidized terpenoids isolated from cellular slime molds. Mucoroidiol showed moderate osteoclast-differentiation inhibitory activity despite demonstrating very weak cell-proliferation inhibitory activity. Therefore, cellular slime molds produce considerably diverse secondary metabolites, and they are promising sources of new natural product chemistry.

Halogen-Substituted Derivatives of Dictyostelium Differentiation-Inducing Factor-1 Suppress Serum-Induced Cell Migration of Human Breast Cancer MDA-MB-231 Cells in Vitro.

Triple-negative breast cancer (TNBC) is highly proliferative and metastatic, and because it lacks three major molecular targets for chemotherapy (estrogen receptor, progesterone receptor, and human epidermal receptor 2), it is extremely refractory. Differentiation-inducing factor 1 (DIF-1) and DIF-3, which are chlorinated alkylphenones, are lead anticancer compounds found in the cellular slime mold Dictyostelium discoideum. Here, we examined the in vitro effects of DIF-1, DIF-3, and 25 DIF derivatives on cell proliferation and serum-induced cell migration in human MDA-MB-231 cells, a model TNBC cell line. We found that Br-DIF-1, a chlorine-to-bromine-substituted derivative of DIF-1, strongly suppressed cell migration (IC50, 3.8 M) with negligible effects on cell proliferation (IC50, >20 M). We then synthesized 18 derivatives of Br-DIF-1 and examined the in vitro effects of these derivatives on cell proliferation and serum-induced cell migration in MDA-MB-231 cells. Among the derivatives, Br-DIF-1(+1), Br-DIF-1(+2), and Br-DIF-3(+2) exhibited strong anti-cell migration activities with IC50 values of 1.5, 1.0, and 3.1 M, respectively, without affecting cell proliferation (IC50, >20 M). These results suggest that these Br-DIF derivatives are good lead compounds for the development of anti-metastatic drugs against TNBC.

Antimicrobial Activities of Dictyostelium Differentiation-Inducing Factors and Their Derivatives.

At the end of its life cycle, the cellular slime mold Dictyostelium discoideum forms a fruiting body consisting of spores and a multicellular stalk. Originally, the chlorinated alkylphenone differentiation-inducing factors (DIFs) -1 and -3 were isolated as stalk cell inducers in D. discoideum. Later, DIFs and their derivatives were shown to possess several biologic activities including antitumor and anti-Trypanosoma properties. In this study, we examined the antibacterial activities of approximately 30 DIF derivatives by using several bacterial species. Several of the DIF derivatives strongly suppressed the growth of the Gram-positive bacteria Staphylococcus aureus, Bacillus subtilis, and Enterococcus faecalis and Enterococcus faecium, at minimum inhibitory concentrations (MICs) in the sub-micromolar to low-micromolar range. In contrast, none of the DIF derivatives evaluated had any noteworthy effect on the growth of the Gram-negative bacterium Escherichia coli (MIC, >100 µM). Most importantly, several of the DIF derivatives strongly inhibited the growth of methicillin-resistant S. aureus and vancomycin-resistant E. faecalis and E. faecium. Transmission electron microscopy revealed that treatment with DIF derivatives led to the formation of distinct multilayered structures consisting of cell wall or plasma membrane in S. aureus. The present results suggest that DIF derivatives are good lead compounds for developing novel antimicrobials.

Biological Activities of Novel Derivatives of Differentiation-Inducing Factor 3 from Dictyostelium discoideum.

Differentiation-inducing factor-3 (DIF-3; 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)hexan-1-one), which is found in the cellular slime mold Dictyostelium discoideum, is a potential candidate compound for the development of new medicines; DIF-3 and its derivatives possess several beneficial biological activities, including anti-tumor, anti-Trypanosoma cruzi, and immunoregulatory effects. To assess the relationship between the biological activities of DIF-3 and its chemical structure, particularly in regard to its alkoxy group and the length of the alkyl chains at the acyl group, we synthesized two derivatives of DIF-3, 1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)octan-1-one (DIF-3(+3)) and 1-(3-chloro-2,6-dihydroxy-4-butoxyphenyl)-hexan-1-one (Hex-DIF-3), and investigated their biological activities in vitro. At micro-molar levels, DIF-3(+3) and Hex-DIF-3 exhibited strong anti-proliferative effects in tumor cell cultures, but their anti-T. cruzi activities at 1 µM in vitro were not as strong as those of other known DIF derivatives. In addition, Hex-DIF-3 at 5 µM significantly suppressed mitogen-induced interleukin-2 production in vitro in Jurkat T cells. These results suggest that DIF-3(+3) and Hex-DIF-3 are promising leads for the development of anti-cancer and immunosuppressive agents.

Activation of cytosolic Slingshot-1 phosphatase by gelsolin-generated soluble actin filaments.

Slingshot-1 (SSH1) is a protein phosphatase that dephosphorylates and activates cofilin, an actin-severing and -disassembling protein. SSH1 is bound to and activated by F-actin, but not G-actin. SSH1 is accumulated in the F-actin-rich lamellipodium but is also diffusely distributed in the cytoplasm. It remains unknown whether SSH1 is activated by soluble (low-level polymerized) actin filaments in the cytoplasm. In this study, we show that SSH1 binds to gelsolin via actin filaments in the cytosolic fraction. Gelsolin promoted solubilization of actin filaments and SSH1 in cell-free assays and in cultured cells. SSH1 was activated by gelsolin-generated soluble actin filaments. Furthermore, gelsolin enhanced cofilin dephosphorylation in neuregulin-stimulated cells. Our results suggest that cytosolic SSH1 forms a complex with gelsolin via soluble actin filaments and is activated by gelsolin-generated soluble actin filaments and that gelsolin promotes stimulus-induced cofilin dephosphorylation through increasing soluble actin filaments, which support SSH1 activation in the cytoplasm.

HTLV-1 induces a Th1-like state in CD4+CCR4+ T cells.

Human T-lymphotropic virus type 1 (HTLV-1) is linked to multiple diseases, including the neuroinflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T cell leukemia/lymphoma. Evidence suggests that HTLV-1, via the viral protein Tax, exploits CD4+ T cell plasticity and induces transcriptional changes in infected T cells that cause suppressive CD4+CD25+CCR4+ Tregs to lose expression of the transcription factor FOXP3 and produce IFN-γ, thus promoting inflammation. We hypothesized that transformation of HTLV-1-infected CCR4+ T cells into Th1-like cells plays a key role in the pathogenesis of HAM/TSP. Here, using patient cells and cell lines, we demonstrated that Tax, in cooperation with specificity protein 1 (Sp1), boosts expression of the Th1 master regulator T box transcription factor (T-bet) and consequently promotes production of IFN-γ. Evaluation of CSF and spinal cord lesions of HAM/TSP patients revealed the presence of abundant CD4+CCR4+ T cells that coexpressed the Th1 marker CXCR3 and produced T-bet and IFN-γ. Finally, treatment of isolated PBMCs and CNS cells from HAM/TSP patients with an antibody that targets CCR4+ T cells and induces cytotoxicity in these cells reduced both viral load and IFN-γ production, which suggests that targeting CCR4+ T cells may be a viable treatment option for HAM/TSP.

Manganese promotes phorbol ester-induced interleukin-2 production via AP-1 activation in Jurkat T-cells.

Mn²âº is a minor nutrient, but is essential for numerous enzymatic activities and thus, for many cellular functions. However, its physiological roles and toxicity remain to be elucidated. In this study, we assessed the pharmacological potential and toxicity of Mn²âº in the immune system by examining the effects of Mn²âº on interleukin-2 (IL-2) production by Jurkat T-cells. Mn²âº at 0.1-1 mM did not significantly induce IL-2 production, whereas phorbol 12-myristate 13-acetate (PMA) at 1 µM slightly induced IL-2 production. Interestingly, Mn²âº at 0.3-0.7 mM promoted PMA-induced IL-2 production in a dose-dependent manner. A reporter gene assay revealed that Mn²âº promoted the activity of AP-1 (activator protein-1, a complex of c-Fos and c-Jun) in the presence of PMA. Western blot analysis showed that Mn²âº promoted the activation of JNK2 (c-Jun N-terminal kinase 2) and p38 MAPK (mitogen-activated protein kinase), which are both activators of AP-1, and upregulated the production of c-Fos and c-Jun within 4h in the presence of PMA. These results suggest that Mn²âº promotes PMA-induced IL-2 production by inducing the production and activation of AP-1, at least in part.

Fucoidan therapy decreases the proviral load in patients with human T-lymphotropic virus type-1-associated neurological disease.

BACKGROUND: Human T-lymphotropic virus type-1 (HTLV-1) is a human retrovirus that causes HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and adult T-cell leukaemia (ATL). A higher viral load in individuals with HTLV-1 infection increases their risk of developing HAM/TSP and ATL. Moreover, the high proviral load is associated with the clinical progression of HAM/TSP. Reduction of the number of HTLV-1-infected cells is therefore crucial for preventing and treating HTLV-1-associated diseases. Recently, fucoidan, a complex sulphated polysaccharide derived from marine seaweed, has been demonstrated to exert inhibitory effects on HTLV-1 infection in vitro. In this study, we examined the in vivo effects of fucoidan on HTLV-1 infection. METHODS: In this single-centre open-label trial, 13 patients with HAM/TSP were treated with 6 g fucoidan daily for 6-13 months. The HTLV-1 proviral DNA load and frequencies of HTLV-1-specific CD8(+) T-cells, natural killer cells, invariant natural killer T-cells and dendritic cells in the peripheral blood were analysed. Furthermore, the in vitro inhibitory effect of fucoidan on cell-to-cell HTLV-1 infection was examined by using luciferase reporter cell assays. RESULTS: Fucoidan inhibited the cell-to-cell transmission of HTLV-1 in vitro. Furthermore, fucoidan therapy resulted in a 42.4% decrease in the HTLV-1 proviral load without affecting the host immune cells. During the treatment, no exacerbation was observed. Four patients with HAM/TSP developed diarrhoea, which improved immediately after stopping fucoidan administration. CONCLUSIONS: Fucoidan is a new potential therapeutic agent for the prevention and treatment of HTLV-1-associated diseases.

Effects of ouabain on contractions induced by manganese ions in C2+a-free, isotonic solutions with varying concentrations of K+ in guinea-pig taenia coli.

The action of ouabain, a cell membrane Na+, K+-ATPase blocker, on contractions induced by manganese ions (Mn2+) in Ca2+-free, isotonic solutions with varying concentrations of K+ in the external medium were investigated in order to evaluate the underlying role of external Na+ in Mn2+-induced contractions in isolated taenia coli of the guinea-pig. Mn2+ at 5 mM induced greater contractions as external isotonic K+ concentrations progressively increased from 10 to 100 mM. Ouabain (2 x 10(-4) M) completely inhibited tension development stimulated by 5 mM Mn2+ in isotonic, 30 mM K+ (96 mM Na+) medium. Whereas, the tension inhibitory effects of ouabain became progressively weaker as isotonic, external K+ concentrations increased to 60 mM, which successively decreased external Na+ concentrations. Eventually, ouabain failed to affect contractions stimulated by Mn2+ in isotonic, 126 mM K+, Na+-deficient medium. Ouabain caused progressively greater increase in cellular Na+ concentrations as the Na+ concentrations increased in the isotonic, K+ medium. While, pyruvate, which penetrates cell independently of external Na+, reversed the inhibition of tension by ouabain in isotonic, 30 mM K+, Na+-sufficient (96 mM) medium containing 5 mM Mn2+. These results suggested that Mn2+ induced the contraction, which was maintained by glucose transport depending on external Na+, in the case of Na+-sufficient medium in K+-depolarized taenia coli. However, it induced the contraction independent of external Na+, in the case of Na+-deficient, K+ medium. Ouabain might exhibit greater inhibition of the contraction induced by Mn2+ as the decrease in the Na+ gradient across the cell membranes continues.

Calmodulin-dependent cyclic nucleotide phosphodiesterase (PDE1) is a pharmacological target of differentiation-inducing factor-1, an antitumor agent isolated from Dictyostelium.

The differentiation-inducing factor-1 (DIF-1) isolated from Dictyostelium discoideum is a potent antiproliferative agent that induces growth arrest and differentiation in mammalian cells in vitro. However, the specific target molecule(s) of DIF-1 has not been identified. In this study, we have tried to identify the target molecule(s) of DIF-1 in mammalian cells, examining the effects of DIF-1 and its analogs on the activity of some candidate enzymes. DIF-1 at 10-40 micro M dose-dependently suppressed cell growth and increased the intracellular cyclic AMP concentration in K562 leukemia cells. It was then found that DIF-1 at 0.5-20 micro M inhibited the calmodulin (CaM)-dependent cyclic nucleotide phosphodiesterase (PDE1) in vitro in a dose-dependent manner. Kinetic analysis revealed that DIF-1 acted as a competitive inhibitor of PDE1 versus the substrate cyclic AMP. Because DIF-1 did not significantly affect the activity of other PDEs or CaM-dependent enzymes and, in addition, an isomer of DIF-1 was a less potent inhibitor, we have concluded that PDE1 is a pharmacological and specific target of DIF-1.

Zinc inhibits calcineurin activity in vitro by competing with nickel.

Calcineurin (CN) is a Ca(2+)/calmodulin (CaM)-dependent protein serine/threonine phosphatase that contains Zn(2+) in its catalytic domain and can be stimulated by divalent ions such as Mn(2+) and Ni(2+). In this study, the role of exogenous Zn(2+) in the regulation of CN activity and its relevance to the role of Ni(2+) was investigated. Zn(2+) at a concentration range of 10nM-10 micro M inhibited Ni(2+)-stimulated CN-activity in vitro in a dose-dependent manner and approximately 50% inhibition was attained with 0.25 micro M Zn(2+). Kinetic analysis showed that Zn(2+) inhibited the activity of CN by competing with Ni(2+). Interaction of CN and CaM was not inhibited with Zn(2+) at 10 micro M. Zn(2+) never affected the activity of cAMP phosphodiesterase 1 or myosin light-chain kinase (CaM-dependent enzymes) and rather activated alkaline phosphatase. The present results indicate that Zn(2+) should be a potent inhibitor for CN activity although this ion is essential for CN.