Identification and characterization of novel components of a Ca2+/calmodulin-dependent protein kinase cascade in HeLa cells

AbstractIn this report, we cloned a novel calmodulin-kinase (CaM-KIδ) from HeLa cells and characterized its activation mechanism. CaM-KIδ exhibits Ca2+/CaM-dependent activity that is enhanced (∼30-fold) in vitro by phosphorylation of its Thr180 by CaM-K kinase (CaM-KK)α, consistent with detection of CaM-KIδ-activating activity in HeLa cells. We also identified a novel CaM-KKβ isoform (CaM-KKβ-3) in HeLa cells whose activity was highly Ca2+/CaM-independent. Transiently expressed CaM-KIδ exhibited enhanced protein kinase activity in HeLa cells without ionomycin stimulation. This sustained activation of CaM-KIδ was completely abolished by Thr180Ala mutation and inhibited by CaM-KK inhibitor, STO-609, indicating a functional CaM-KK/CaM-KIδ cascade in HeLa cells

B-Cell-Activating Factor Affects the Occurrence of Thyroid Autoimmunity in Chronic Hepatitis C Patients Treated with Interferon Alpha

Chronic hepatitis C (CHC) patients frequently suffer from thyroid disorders during interferon therapy. However, the mechanism remains unclear. In this study, we investigated the association between serum B-cell-activating factor belonging to the TNF family (BAFF) levels and the presence of antithyroid peroxidase antibody (anti-TPO) in CHC patients treated with pegylated interferon alpha and ribavirin combination therapy. Six months after the therapy, anti-TPO antibody was detected in 10 (males, 1; females, 9) of 50 patients. The mean age of these patients was higher than that of the anti-TPO-negative patients (61 yr versus 55 yr). Before treatment, the serum BAFF levels of the anti-TPO-positive patients were higher than those of the anti-TPO-negative patients. After starting therapy, the serum BAFF levels of both the anti-TPO-positive and -negative patient groups were elevated. Our findings suggest that the serum BAFF concentration before therapy can predict the risk of thyroid autoimmunity in elderly female patients with CHC

Phosphorylation of myosin II regulatory light chain controls its accumulation, not that of actin, at the contractile ring in HeLa cells

During cytokinesis in eukaryotic cells, an actomyosin-based contractile ring (CR) is assembled along the equator of the cell. Myosin II ATPase activity is stimulated by the phosphorylation of the myosin II regulatory light chain (MRLC) in vitro, and phosphorylated MRLC localizes at the CR in various types of cells. Previous studies have determined that phosphorylated MRLC plays an important role in CR furrowing. However, the role of phosphorylated MRLC in CR assembly remains unknown. Here, we have used confocal microscopy to observe dividing HeLa cells expressing fluorescent protein-tagged MRLC mutants and actin during CR assembly near the cortex. Di-phosphomimic MRLC accumulated at the cell equator earlier than non-phosphorylatable MRLC and actin. Interestingly, perturbation of myosin II activity by non-phosphorylatable MRLC expression or treatment with blebbistatin, a myosin II inhibitor, did not alter the time of actin accumulation at the cell equator. Furthermore, inhibition of actin polymerization by treatment with latrunculin A had no effect on MRLC accumulation at the cell equator. Taken together, these data suggest that phosphorylated MRLC temporally controls its own accumulation, but not that of actin, in cultured mammalian cells

Acutaxylines A and B, two novel triterpenes from Dysoxylum acutangulum

Two novel triterpenes, acutaxylines A (1) and B (2) consisting of a cyclopentenone side chain at C-17 and an oxepan-2-ol, were isolated from the leaves of Dysoxylum acutangulum. The relative stereochemistry of 1 and 2 was determined by NOESY correlations. Acutaxyline B showed moderate cytotoxicity against human blood premyelocytic leukemia cells

Chrotacumines A-D, chromone alkaloids from Dysoxylum acutangulum

Four new chromone alkaloids, chrotacumines A-D (1-4), consisting of a 5,7-dihydroxy-2-methylchromone, an N-Me piperidine ring, and an ester side chain were isolated from Dysoxylum acutangulum, and their structures including absolute configurations were elucidated on the basis of spectroscopic data interpretation including 2D NMR, CD spectra, and X-ray analysis. The known compound rohitukine (5) showed moderate cytotoxicity against human HL-60 promyelocytic leukemia and HCT-116 colon cancer cells

Serum Reactivity Against Bacterial Pyruvate Dehydrogenase: Increasing the Specificity of Anti-Mitochondrial Antibodies for the Diagnosis of Primary Biliary Cirrhosis

Antimitochondrial antibodies (AMA) are the serum hallmark of primary biliary cirrhosis (PBC). However, AMA-positivity can be found in non-PBC sera when lower dilutions are used, thus raising issues about the specificity and sensitivity of the test. AMA reacts primarily with the lipoylated domains of pyruvate dehydrogenase-E2 (PDC-E2) which is highly conserved across species, including bacteria. We studied 77 serum samples, including 24 from patients with anti-PDC-E2-positive PBC and 53 controls (16 with autoimmune hepatitis (AIH), 10 with primary sclerosing cholangitis (PSC), and 27 healthy individuals) for their reactivities at serial dilutions (1:10, 1:20 and 1:40) against Escherichia coli DH5 alpha lysate overexpressing human PDC-E2 using immunoblotting (IB). A murine anti-human PDC-E2 monoclonal antibody (mAB) was used as control. We further studied positive sera using adsorption with a synthetic E. coli peptide sharing similarity with human PDC-E2. Finally, we verified whether a unique buffer for E. coli preparation could reduce non-specific serum reactivity. Results demonstrated that 100% of anti-PDC-E2-positive PBC and up to 38% of control sera at 1:10 dilution recognized E. coli PDC-E2 at IB while dilution tests indicated that the overall potency of PBC reactivity was 100-fold higher compared to controls. In fact, a subgroup (20-38%) of non-PBC sera were positive at low titers but lost the reactivity when absorbed with the synthetic E. coli peptide. Finally, our unique buffer reduced the reactivity of non-PBC sera as measured by ELISA. In conclusion, we demonstrated that weak cross-reactivity with E. coli PDC-E2 occurs in non-PBC sera at lower dilutions and that such reactivity is not due to AMA-positivity. The use of a specific buffer might avoid the risk of false positive AMA determinations when E. coli-expressed recombinant antigens are used

Esophageal transit study using a sliding sum image: application to patients with probable and definite systemic sclerosis

金沢大学医薬保健研究域医学系Purpose: Esophageal complication is common in systemic sclerosis (SSc), but scintigraphic transit patterns based on each subtype have not been understood well. The aim of this study was to develop a new algorithm for integrating a dynamic esophageal transit study and to apply the method to patients with SSc. Methods: A total of 40 patients suspected of having SSc were examined by a dynamic esophageal transit study. The subtypes included 32 with definite SSc (15 limited cutaneous type and 17 diffuse cutaneous type) and 8 with probable SSc. The serial esophageal images were shifted and summed to a functional image (sliding sum image) and compared to a conventional condensed image analysis. Esophageal retention fraction at 90 s (R90) and half-time (T1/2) of transit were also measured. Results: The four patterns of the sliding sum image and condensed image agreed in all patients. Abnormal retention patterns were observed in none of the 8 (0%) patients with the probable SSc and in 15 of 32 (47%) patients with definite SSc (p = 0.014). The severity of scleroderma assessed by modified Rodnan skin thickness score correlated with that of esophageal retention R90 (p = 0.04). Conclusion: The sliding sum image is a simple and effective method for integrating esophageal transit. Patients with definite SSc and severe scleroderma had significantly higher retention patterns, while probable SSc patients showed no esophageal dysmotility. © 2011 The Japanese Society of Nuclear Medicine

Microplastic Beads Incorporated into a Single Cell ： Analyses Using the Green Paramecium, Paramecium bursaria

The unicellular protist Paramecium bursaria harbors hundreds of symbiotic algae resembling Chlorella species in the cell. It is thought that the host P. bursaria uses some of photosynthetic products from these algae when sunlight is available. When photosynthesis cannot be performed, P. bursaria preys on bacteria, molds, algae, etc. in the surroundings for an energy source. Interestingly, some of the algae were observed to move from the food vacuole to the cytoplasm, becoming symbionts, within several days after incorporation into the cell. Since P. bursaria is benthic, it should be possible for various kinds of precipitated tiny particles to be taken up into the cell body during predation. In this study, microplastic (MP) beads with a diameter of 1 μm, which is about the same size as the algae, were mixed in the suspension medium of P. bursaria . It was observed that uptake of the beads into the cell body of P. bursaria started within 5 min after mixing, and the beads were observed inside P. bursaria even several days after the addition to the P. bursaria culture suspension. It is highly probable that the MP beads observed in the cell body somehow escaped from the food vacuole and moved into the cytoplasm.Full-Length PaperBy a grant from Research Institute for Integrated Science, Kanagawa Universit