The binding of ATP to the catalytic and the regulatory site of Ca2+,Mg2+-dependent ATPase of the sarcoplasmic reticulum

Two kinds of ATP binding sites were found on the ATPase molecule in deoxycholic acid-treated sarcoplasmic reticulum. One was the catalytic site (1 mol/mol active site) and its affinity was high. Upon addition of Ca²⁺, all the ATP bound to the catalytic site disappeared at 75 mM KCl, while a significant amount of ATP remained bound to the site at 0–2 mM KCl. The latter binding was found to be due to the formation of a slowly exchanging enzyme-ATP complex, which is in equilibrium with phosphoenzyme + ADP. The other binding site was the regulatory one (1 mol/mol active site) and its affinity was low, changing only insignificantly upon addition of Ca²⁺. The ATP binding to the regulatory site shifted the equilibrium between the slowly exchanging complex and EP toward EP.     

An anchorage-dependent locus in the cell cycle for the growth of 3T3 cells

Mouse fibroblasts, 3T3 cells, require a solid surface for continuous growth, but when 3T3 cells, during their exponential phase in Petri dishes, were transferred to a suspension culture, the number of cells roughly doubled by 30 h. During the suspension culture the number of pairing cells (c₂) increased, but that of the single cells decreased. When cells synchronized at mitosis or at the G1-S boundary were transferred to the suspension culture, the number of pairing cells peaked at 30 min and at 10 h, respectively. DNA synthesis began immediately after the cells, which were cultured for 16 h in the suspension, had settled onto the surface of the Petri dishes. When cells in a confluent culture were arrested at an early G1 period and were suspended, the number of pairing cells did not increase. These results indicate that the most important locus for anchorage growth seems to be at a late G1 period of the cell cycle.     

Metabolism of PG in man: Effect of furosemide on the excretion of the main metabolite of PG F2α

The excretion rates of main urinary metabolite of PG F₂α were measured radioimmunologically in 4 healthy persons and in 13 essential hypertensives. The resting values were 9.3±0.73 in the former and 10.4±2.17 ng/min in the latter. There was no significant differences between them. The excretion of the metabolite decresed prominently after the administration of furosemide. The percent decrease was 57% in healthy persons and 70% in essential hypertension. The percent result supports that furosemide inhibit the catabolism of PG F₂α.     

Grayanoside C,a new diterpene glucoside from Leucothoe grayana

A new diterpene glucoside has been isolated from Leucothoe grayana. Its structure was elucidated by X-ray diffraction analysis of a dehydrated product of the aglycone and conversion of the same to leucothol A.     

Hermaphroditism in fishes: an annotated list of species,phylogeny, and mating system

Fewer than 1% of vertebrate species are hermaphroditic, and essentially all of these are fishes. Four types of hermaphroditism are known in fishes: simultaneous (or synchronous) hermaphroditism (SH), protandry (male-to-female sex change; PA), protogyny (female-to-male sex change; PG), and bidirectional sex change (BS or reversed sex change in protogynous species). Here we present an annotated list of hermaphroditic fish species from a comprehensive review and careful re-examination of all primary literature. We confirmed functional hermaphroditism in more than 450 species in 41 families of 17 teleost orders. PG is the most abundant type (305 species of 20 families), and the others are much less abundant, BS in 66 species of seven families, SH in 55 species of 13 families, and PA in 54 species of 14 families. The recently proposed phylogenetic tree indicated that SH and PA have evolved several times in not-closely related lineages of Teleostei but that PG (and BS) has evolved only in four lineages of Percomorpha. Examination of the relation between hermaphroditism type and mating system in each species mostly supported the size-advantage model that predicts the evolution of sequential hermaphroditism. Finally, intraspecific variations in sexual pattern are discussed in relation to population density, which may cause variation in mating system.

     

Arabidopsis Raf‐like kinases act as positive regulators of subclass III SnRK2 in osmostress signaling

Given their sessile nature, land plants must use various mechanisms to manage dehydration under water‐deficit conditions. Osmostress‐induced activation of the SNF1‐related protein kinase 2 (SnRK2) family elicits physiological responses such as stomatal closure to protect plants during drought conditions. With the plant hormone ABA receptors [PYR (pyrabactin resistance)/PYL (pyrabactin resistance‐like)/RCAR (regulatory component of ABA receptors) proteins] and group A protein phosphatases, subclass III SnRK2 also constitutes a core signaling module for ABA, and osmostress triggers ABA accumulation. How SnRK2 is activated through ABA has been clarified, although its activation through osmostress remains unclear. Here, we show that Arabidopsis ABA and abiotic stress‐responsive Raf‐like kinases (AtARKs) of the B3 clade of the mitogen‐activated kinase kinase kinase (MAPKKK) family are crucial in SnRK2‐mediated osmostress responses. Disruption of AtARKs in Arabidopsis results in increased water loss from detached leaves because of impaired stomatal closure in response to osmostress. Our findings obtained in vitro and in planta have shown that AtARKs interact physically with SRK2E, a core factor for stomatal closure in response to drought. Furthermore, we show that AtARK phosphorylates S171 and S175 in the activation loop of SRK2E in vitro and that Atark mutants have defects in osmostress‐induced subclass III SnRK2 activity. Our findings identify a specific type of B3‐MAPKKKs as upstream kinases of subclass III SnRK2 in Arabidopsis. Taken together with earlier reports that ARK is an upstream kinase of SnRK2 in moss, an existing member of a basal land plant lineage, we propose that ARK/SnRK2 module is evolutionarily conserved across 400 million years of land plant evolution for conferring protection against drought.     

Different effects of mating group size as male and as female on sex allocation in a simultaneous hermaphrodite

Sex allocation theory predicts that the optimal sexual resource allocation of simultaneous hermaphrodites is affected by mating group size (MGS). Although the original concept assumes that the MGS does not differ between male and female functions, the MGS in the male function (MGSm; i.e., the number of sperm recipients the focal individual can deliver its sperm to plus one) and that in the female function (MGSf; the number of sperm donors plus one) do not always coincide and may differently affect the optimal sex allocation. Moreover, reproductive costs can be split into “variable” (e.g., sperm and eggs) and “fixed” (e.g., genitalia) costs, but these have been seldom distinguished in empirical studies. We examined the effects of MGSm and MGSf on the fixed and variable reproductive investments in the sessilian barnacle Balanus rostratus. The results showed that MGSm had a positive effect on sex allocation, whereas MGSf had a nearly significant negative effect. Moreover, the “fixed” cost varied with body size and both aspects of MGS. We argue that the two aspects of MGS should be distinguished for organisms with unilateral mating.     

Successful adjuvant bi-weekly gemcitabine chemotherapy for pancreatic cancer without impairing patients’ quality of life

Background

Although adjuvant gemcitabine (GEM) chemotherapy for pancreatic cancer is standard, the quality of life (QOL) in those patients is still impaired by the standard regimen of GEM. Therefore, we studied whether mild dose-intensity adjuvant chemotherapy with bi-weekly GEM administration could provide a survival benefit with acceptable QOL to the patients with pancreatic cancer.

Methods

After a phase I trial, an adjuvant bi-weekly 1,000 mg/m² of GEM chemotherapy was performed in 58 patients with pancreatic cancer for at least 12 courses (Group A). In contrast, 36 patients who declined the adjuvant bi-weekly GEM chemotherapy underwent traditional adjuvant 5FU-based chemotherapy (Group B). Careful periodical follow-ups for side effects of GEM and disease recurrence, and assessment of patients’ QOL using the EORTC QOL questionnaire (QLQ-C30) and pancreatic cancer-specific supplemental module (QLQ-PAN26) were performed. Retrospectively, the degree of side effects, patients’ QOL, compliance rate, disease-free survival (DFS), and overall survival (OS) in Group A were compared with those in Group B.

Results

No severe side effects (higher than Grade 2 according to the common toxicity criteria of ECOG) were observed, except for patients in Group B, who were switched to the standard GEM chemotherapy. Patients’ QOL was better in Group A than B (fatigue: 48.9 ± 32.1 versus 68.1 ± 36.3, nausea and vomiting: 26.8 ± 20.4 versus 53.7 ± 32.6, diarrhea: 21.0 ± 22.6 versus 53.9 ± 38.5, difficulty gaining weight: 49.5 ± 34.4 versus 67.7 ± 40.5, P < 0.05). Compliance rates in Groups A and B were 93% and 47%. There was a significant difference in the median DFS between both groups (Group A : B =12.5 : 6.6 months, P < 0.001). The median OS of Group A was prolonged markedly compared with Group B (20.2 versus 11.9 months, P < 0.005). For OS between both groups, univariate analysis revealed no statistical difference in 69-year-old or under females, and T1–2 factors, moreover, multivariate analysis indicated three factors, such as bi-weekly adjuvant GEM chemotherapy, T2 or less, and R0.

Conclusions

Adjuvant chemotherapy with bi-weekly GEM offered not only the advantage of survival benefits but the excellent compliance with acceptable QOL for postoperative pancreatic cancer patients.     

Vitamin D2 interacts with Human PrPc (90–231) and breaks PrPc oligomerization in vitro

PrP^sc, the pathogenic isoform of PrP^c, can convert PrP^c into PrP^sc through direct interactions. PrP^c oligomerization is a required processing step before PrP^sc formation, and soluble oligomers appear to be the toxic species in amyloid-related disorders. In the current study, direct interactions between vitamin D₂ and human recombinant PrP^c (90–231) were observed by Biacore assay, and 3F4 antibody, specific for amino acid fragment 109–112 of PrP^c, inhibited this interaction. An ELISA study using3F4 antibody showed that PrP^c (101–130), corresponding sequence to human PrP, was affected by vitamin D₂, supporting the results of Biacore studies and suggesting that the PrP^c sequence around the 3F4 epitope was responsible for the interaction with vitamin D₂. Furthermore, the effects of vitamin D₂ on disruption of PrP^c (90–231) oligomerization were elucidated by dot blot analysis and differential protease k susceptibilities. While many chemical compounds have been proposed as potential therapeutic agents for the treatment of scrapie, most of these are toxic. However, given the safety and blood brain barrier permeability of vitamin D₂, we propose that vitamin D₂ may be a suitable agent to target PrP^c in the brain and therefore is a potential therapeutic candidate for prion disease.