Steady-state force–velocity relation of ATP-dependent sliding between slime mold myosin, arranged on paramyosin filaments, and algal cell actin cables

To investigate characteristics of ATP-dependent sliding of a non-muscle cell myosin, obtained from a cellular slime mold Dictyostelium discoideum, on actin filament, we prepared hybrid thick filaments, in which Dictyostelium myosin was regularly arranged around paramyosin filaments obtained from a molluscan smooth muscle. A single to a few hybrid filaments were attached to a polystyrene bead (diameter, 4.5 μm; specific gravity, 1.5), and the filaments were made to slide on actin filament arrays (actin cables) in the internodal cell of an alga Chara corallina, mounted on the rotor of a centrifuge microscope. The filament-attached bead was observed to move with a constant velocity under a constant external load for many seconds. The steady-state force–velocity relation of Dictyostelium myosin sliding on actin cables was hyperbolic in shape except for large loads ≤0.7–0.8 P₀, being qualitatively similar to that of skeletal muscle fibres, despite a considerable variation in the number of myosin molecules interacting with actin cables. Comparison of the P–V curves between Dictyostelium myosin and muscle myosins sliding on actin cables suggests that the time of attachment to actin in a single attachment–detachment cycle is much longer in Dictyostelium myosin than in muscle myosins.     

Age-related changes of element contents in the human meniscus

The relative contents (RCs) of elements in the human menisci from 23 subjects in the age range between 65 and 93 yr were analyzed by inductively coupled plasma atomic emission spectrometry. The RCs of sulfur, calcium, and phosphorus in menisci increased progressively until the 80s, being the highest in the 80s, and thereafter decreased. The RCs of magnesium in menisci increased progressively until the 90s. Regarding the medial and lateral menisci, higher RCs of magnesium and iron, and a lower RC of phosphorus were found in lateral menisci in comparison with those in medial menisci. There were sexual differences in the RCs of calcium and phosphorus of medial and lateral menisci. The RCs of calcium and phosphorus were about 50% higher in women’s menisci than in men’s. Histological examinations showed that structureless mucoid masses were observed in the menisci, with very high RCs of calcium and phosphorus being detected.     

Effects of Ovariectomy and Prolactin on Mammary Gland Expressions of Transforming Growth Factor alpha and Epidermal Growth Factor Receptor mRNAs in Mice

Beginning 15 days after ovariectomy (OVX), a high mammary tumor strain of SHN virgin mice at 3 months of age received subcutaneous injections of danazol (0.5 mug / 0.1 ml olive oil, once a day), perphenazine (0.05 mg / 0.1 ml saline, twice a day) or ovine prolactin (oPRL: 0.25 mg / 0.05 ml buffer, twice a day) for 3 days to modulate their circulating PRL levels. The serum PRL level was significantly decreased by danazol and increased by perphenazine compared to the intact and OVX-control groups. The expression of both transforming growth factor alpha (TGFalpha) mRNA and epidermal growth factor receptor (EGFR) mRNA in the mammary gland was increased by danazol. However, TGFalpha mRNA expression was decreased by perphenazine. Meanwhile, mammary end-bud formation was inhibited in danazol-treated group. All findings suggest that the manifestation of the effect of TGFalpha on mammary gland is rather suppressed by PRL, while mammary gland growth needs the participation of PRL; in other words, PRL is dominant to TGFalpha on the mammary gland growth. OVX resulted in a significant decrease of TGFalpha mRNA expression in the mammary gland despite of little alteration in serum PRL, confirming the previous observations. The similar trend was observed in ICR mice; however, the response to hormonal modulation is generally less susceptible than SHN mice.     

Unrestricted modification search reveals lysine methylation as major modification induced by tissue formalin fixation and paraffin embedding

Formalin‐fixed paraffin‐embedded (FFPE) tissue is considered as an appropriate alternative to frozen/fresh tissue for proteomic analysis. Here we study formalin‐induced alternations on a proteome‐wide level. We compared LC‐MS/MS data of FFPE and frozen human kidney tissues by two methods. First, clustering analysis revealed that the biological variation is higher than the variation introduced by the two sample processing techniques and clusters formed in accordance with the biological tissue origin and not with the sample preservation method. Second, we combined open modification search and spectral counting to find modifications that are more abundant in FFPE samples compared to frozen samples. This analysis revealed lysine methylation (+14 Da) as the most frequent modification induced by FFPE preservation. We also detected a slight increase in methylene (+12 Da) and methylol (+30 Da) adducts as well as a putative modification of +58 Da, but they contribute less to the overall modification count. Subsequent SEQUEST analysis and X!Tandem searches of different datasets confirmed these trends. However, the modifications due to FFPE sample processing are a minor disturbance affecting 2–6% of all peptide‐spectrum matches and the peptides lists identified in FFPE and frozen tissues are still highly similar.     

Treatment of Irradiated Mice with High-Dose Ascorbic Acid Reduced Lethality

Ascorbic acid is an effective antioxidant and free radical scavenger. Therefore, it is expected that ascorbic acid should act as a radioprotectant. We investigated the effects of post-radiation treatment with ascorbic acid on mouse survival. Mice received whole body irradiation (WBI) followed by intraperitoneal administration of ascorbic acid. Administration of 3 g/kg of ascorbic acid immediately after exposure significantly increased mouse survival after WBI at 7 to 8 Gy. However, administration of less than 3 g/kg of ascorbic acid was ineffective, and 4 or more g/kg was harmful to the mice. Post-exposure treatment with 3 g/kg of ascorbic acid reduced radiation-induced apoptosis in bone marrow cells and restored hematopoietic function. Treatment with ascorbic acid (3 g/kg) up to 24 h (1, 6, 12, or 24 h) after WBI at 7.5 Gy effectively improved mouse survival; however, treatments beyond 36 h were ineffective. Two treatments with ascorbic acid (1.5 g/kg × 2, immediately and 24 h after radiation, 3 g/kg in total) also improved mouse survival after WBI at 7.5 Gy, accompanied with suppression of radiation-induced free radical metabolites. In conclusion, administration of high-dose ascorbic acid might reduce radiation lethality in mice even after exposure.     

Function and Evolutionary Origin of Unicellular Camera-Type Eye Structure

The ocelloid is an extraordinary eyespot organelle found only in the dinoflagellate family Warnowiaceae. It contains retina- and lens-like structures called the retinal body and the hyalosome. The ocelloid has been an evolutionary enigma because of its remarkable resemblance to the multicellular camera-type eye. To determine if the ocelloid is functionally photoreceptive, we investigated the warnowiid dinoflagellate Erythropsidinium. Here, we show that the morphology of the retinal body changed depending on different illumination conditions and the hyalosome manifests the refractile nature. Identifying a rhodopsin gene fragment in Erythropsidinium ESTs that is expressed in the retinal body by in situ hybridization, we also show that ocelloids are actually light sensitive photoreceptors. The rhodopsin gene identified is most closely related to bacterial rhodopsins. Taken together, we suggest that the ocelloid is an intracellular camera-type eye, which might be originated from endosymbiotic origin.     

Integrated Copy Number and Expression Analysis Identifies Profiles of Whole-Arm Chromosomal Alterations and Subgroups with Favorable Outcome in Ovarian Clear Cell Carcinomas

Ovarian clear cell carcinoma (CCC) is generally associated with chemoresistance and poor clinical outcome, even with early diagnosis; whereas high-grade serous carcinomas (SCs) and endometrioid carcinomas (ECs) are commonly chemosensitive at advanced stages. Although an integrated genomic analysis of SC has been performed, conclusive views on copy number and expression profiles for CCC are still limited. In this study, we performed single nucleotide polymorphism analysis with 57 epithelial ovarian cancers (31 CCCs, 14 SCs, and 12 ECs) and microarray expression analysis with 55 cancers (25 CCCs, 16 SCs, and 14 ECs). We then evaluated PIK3CA mutations and ARID1A expression in CCCs. SNP array analysis classified 13% of CCCs into a cluster with high frequency and focal range of copy number alterations (CNAs), significantly lower than for SCs (93%, P < 0.01) and ECs (50%, P = 0.017). The ratio of whole-arm to all CNAs was higher in CCCs (46.9%) than SCs (21.7%; P < 0.0001). SCs with loss of heterozygosity (LOH) of BRCA1 (85%) also had LOH of NF1 and TP53, and LOH of BRCA2 (62%) coexisted with LOH of RB1 and TP53. Microarray analysis classified CCCs into three clusters. One cluster (CCC-2, n = 10) showed more favorable prognosis than the CCC-1 and CCC-3 clusters (P = 0.041). Coexistent alterations of PIK3CA and ARID1A were more common in CCC-1 and CCC-3 (7/11, 64%) than in CCC-2 (0/10, 0%; P < 0.01). Being in cluster CCC-2 was an independent favorable prognostic factor in CCC. In conclusion, CCC was characterized by a high ratio of whole-arm CNAs; whereas CNAs in SC were mainly focal, but preferentially caused LOH of well-known tumor suppressor genes. As such, expression profiles might be useful for sub-classification of CCC, and might provide useful information on prognosis.