Prognostic Value of Radiological Response to Chemotherapy in Patients with Osteosarcoma

Background

Chemotherapy is essential to improve the prognosis of the patients with osteosarcoma, and the response to chemotherapy is an important prognostic factor. In this study, the impact of various radiological examinations on overall survival (OS) and event-free survival (EFS) was evaluated.

Method

Eighty-two patients with high-grade osteosarcoma were included in this study, and we evaluated the following factors for prognostic significance: age (≥40 years), gender (male), tumor location (truncal site), metastatic disease, histological response to chemotherapy, radiological response to chemotherapy assessed using X-ray, angiography, CT, MRI, ²⁰¹Tl scintigraphy, and ^99mTc-MIBI scintigraphy (^99mTc-MIBI), and combined radiological score (CRS).

Results

Univariate analyses revealed that metastatic disease, histological response, ^99mTc-MIBI, and CRS were significantly correlated with OS. Multivariate analyses showed that metastatic disease (OS: HR 35.9, P<0.001; EFS: HR 17.32, P<0.001) was an independent predictor of OS and EFS. Tumor location (HR 36.1, P = 0.003), histological response (HR 31.1, P = 0.036), and ^99mTc-MIBI (HR 18.4, P = 0.038) were significant prognostic factors for OS. Moreover, CRS was a marginally significant predictor of OS and EFS.

Conclusion

The chemotherapeutic effects evaluated by ^99mTc-MIBI and CRS could be considered as prognostic factors in osteosarcoma.     

Ca2+-independent Activation of Ca2+/Calmodulin-dependent Protein Kinase II Bound to the C-terminal Domain of CaV2.1 Calcium Channels

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) forms a major component of the postsynaptic density where its functions in synaptic plasticity are well established, but its presynaptic actions are poorly defined. Here we show that CaMKII binds directly to the C-terminal domain of Ca_V2.1 channels. Binding is enhanced by autophosphorylation, and the kinase-channel signaling complex persists after dephosphorylation and removal of the Ca²⁺/CaM stimulus. Autophosphorylated CaMKII can bind the Ca_V2.1 channel and synapsin-1 simultaneously. CaMKII binding to Ca_V2.1 channels induces Ca²⁺-independent activity of the kinase, which phosphorylates the enzyme itself as well as the neuronal substrate synapsin-1. Facilitation and inactivation of Ca_V2.1 channels by binding of Ca²⁺/CaM mediates short term synaptic plasticity in transfected superior cervical ganglion neurons, and these regulatory effects are prevented by a competing peptide and the endogenous brain inhibitor CaMKIIN, which blocks binding of CaMKII to Ca_V2.1 channels. These results define the functional properties of a signaling complex of CaMKII and Ca_V2.1 channels in which both binding partners are persistently activated by their association, and they further suggest that this complex is important in presynaptic terminals in regulating protein phosphorylation and short term synaptic plasticity.     

Queen-worker conflicts over male production and sex allocation in a primitively eusocial wasp

Abstract In a colony headed by a single monandrous foundress, theories predict that conflicts between a queen and her workers over both sex ratio and male production should be intense. If production of males by workers is a function of colony size, this should affect sex ratios, but few studies have examined how queens and workers resolve both conflicts simultaneously. We conducted field and laboratory studies to test whether sex-ratio variation can be explained by conflict over male production between queen and workers in the primitively eusocial wasp Polistes chinensis antennalis.
Worker oviposition rate increased more rapidly with colony size than did queen oviposition. Allozyme and micro-satellite markers revealed that the mean frequency of workers' sons among male adults in queen-right colonies was 0.39 ± 0.08 SE (n = 22). Genetic relatedness among female nestmates was high (0.654–0.796), showing that colonies usually had a single, monandrous queen. The mean sex allocation ratio (male investment/male and gyne investments) of 46 queen-right colonies was 0.47 ± 0.02, and for 25 orphaned colonies was 0.86 ± 0.04. The observed sex allocation ratio was likely to be under queen control. For queen-right colonies, the larger colonies invested more in males and produced reproductives protandrously and/or simultaneously, whereas the smaller colonies invested more in females and produced reproductives protogynously. Instead of positive relationships between colony size and worker oviposition rate, the frequency of workers' sons within queen-right colonies did not increase with colony size. These results suggest that queens control colony investment, even though they allow worker oviposition in queen-right colonies. Eggs laid by workers may be policed by the queen and/or fellow workers. Worker oviposition did not influence the outcome of sex allocation ratio as a straightforward function of colony size.     

Direct Visualization of the Translocation of the γ-Subspecies of Protein Kinase C in Living Cells Using Fusion Proteins with Green Fluorescent Protein

We expressed the γ-subspecies of protein kinase C (γ-PKC) fused with green fluorescent protein (GFP) in various cell lines and observed the movement of this fusion protein in living cells under a confocal laser scanning fluorescent microscope. γ-PKC–GFP fusion protein had enzymological properties very similar to that of native γ-PKC. The fluorescence of γ-PKC– GFP was observed throughout the cytoplasm in transiently transfected COS-7 cells. Stimulation by an active phorbol ester (12-O-tetradecanoylphorbol 13-acetate [TPA]) but not by an inactive phorbol ester (4α-phorbol 12, 13-didecanoate) induced a significant translocation of γ-PKC–GFP from cytoplasm to the plasma membrane. A23187, a Ca²⁺ ionophore, induced a more rapid translocation of γ-PKC–GFP than TPA. The A23187-induced translocation was abolished by elimination of extracellular and intracellular Ca²⁺. TPA- induced translocation of γ-PKC–GFP was unidirected, while Ca²⁺ ionophore–induced translocation was reversible; that is, γ-PKC–GFP translocated to the membrane returned to the cytosol and finally accumulated as patchy dots on the plasma membrane. To investigate the significance of C1 and C2 domains of γ-PKC in translocation, we expressed mutant γ-PKC–GFP fusion protein in which the two cysteine rich regions in the C1 region were disrupted (designated as BS 238) or the C2 region was deleted (BS 239). BS 238 mutant was translocated by Ca²⁺ ionophore but not by TPA. In contrast, BS 239 mutant was translocated by TPA but not by Ca²⁺ ionophore. To examine the translocation of γ-PKC–GFP under physiological conditions, we expressed it in NG-108 cells, N-methyl-d-aspartate (NMDA) receptor–transfected COS-7 cells, or CHO cells expressing metabotropic glutamate receptor 1 (CHO/mGluR1 cells). In NG-108 cells , K⁺ depolarization induced rapid translocation of γ-PKC–GFP. In NMDA receptor–transfected COS-7 cells, application of NMDA plus glycine also translocated γ-PKC–GFP. Furthermore, rapid translocation and sequential retranslocation of γ-PKC–GFP were observed in CHO/ mGluR1 cells on stimulation with the receptor. Neither cytochalasin D nor colchicine affected the translocation of γ-PKC–GFP, indicating that translocation of γ-PKC was independent of actin and microtubule. γ-PKC–GFP fusion protein is a useful tool for investigating the molecular mechanism of γ-PKC translocation and the role of γ-PKC in the central nervous system.Protein kinase C (PKC),¹ a family of phospholipid-dependent serine/threonine kinases and of which there are at least 12 subspecies, plays an important role in various cellular signal transductions (Nishizuka, 1984, 1988, 1992). Regardless of ubiquitous expression of PKCs in various tissues, the central nervous system abundantly contains several unique PKCs. In particular, the γ-subspecies of PKC (γ-PKC) is present only in the central nervous system and is thought to be involved in many neuronal functions including the formation of neural plasticity and memory (Nishizuka, 1986; Abeliovich et al., 1993a ,b; Tanaka and Nishizuka, 1994).PKC isozymes are divided into three subfamilies based on differences in the regulatory domain: conventional PKC (cPKC), novel PKC (nPKC), and atypical PKC (aPKC). Conventional PKCs have two common regions in the regulatory domain, C1 and C2. The C1 region has two cysteine-rich loops (zinc finger–like motifs) that interact with diacylglycerol (DG) or phorbol esters (Nishizuka, 1988; Ono et al., 1989). The C2 region mediates calcium binding (Ono et al., 1989) and is only present in cPKCs (Ono et al., 1988b ), although a region related to C2 region was recently reported in nPKC, a calcium-independent PKC (Parker and Dekker, 1997). Full activation of cPKCs, including γ-PKC, requires DG and calcium. The C1 region is also present in nPKC, and one of the cysteine-rich loops is found in aPKCs.Conventional PKCs and nPKCs, whose regulatory domains contain C1, are known to be translocated from the cytosol to particulate fraction when activated by DG or phorbol esters (Kraft et al., 1982). Therefore, the translocation of PKCs is a good marker of whether these enzymes are activated. Although this phenomenon is well known, the mechanism and physiological significance of PKC translocation have not yet been clarified. By conventional enzymological or immunohistochemical methods, it is impossible to observe the translocation of PKC in real time, in the same cells, and in living states, except in the investigation using fluorescent probes that directly bind PKC (Chen and Poenie, 1993). In addition, these fluorescent compounds are suggested to inhibit the activity of PKC itself at high concentration.To resolve these problems and to directly observe the translocation of γ-PKC in living cells, we produced a fusion protein of γ-PKC and green fluorescent protein (GFP). The GFP, isolated from jellyfish Aequorea victoria, has fluorescence without additional substrates and cofactors (Cubitt et al., 1995). Recent studies have revealed that GFP is a good candidate as a molecular reporter protein to monitor the alternation of protein localization, gene expression, and protein trafficking in living cells (Cubitt et al., 1995). In this study, we visualized and analyzed the translocation of γ-PKC–GFP fusion protein with confocal laser scanning fluorescence microscopy, using various stimulations, such as phorbol esters, Ca²⁺ ionophore, K⁺ depolarization, and receptor-mediated stimulus.     

Effect of season on the fatty acid composition and free amino acid content of the sardine Sardinops melanostictus

The purpose of this study was to clarify the seasonal variation of fatty acid composition and free amino acid content in the Japanese sardine (Sardinops melanostictus) from the sea of Hyuga-Nada, and the relationship between the fatty acid composition of this sardine and that of plankton in the area. The lipid content of sardines at the sea of Hyuga-Nada was low in February (1.8%) and high (7.2%) from July to September. The major fatty acids in the total lipids from sardine were myristic acid (14:0), palmitic acid (16:0), stearic acid (18:0), palmitoleic acid (16:1 n-7), oleic acid (18:1 n-9), eicosapentaenoic acid (20:5 n-3), and docosahexaenoic acid (22:6 n-3). The characteristics of the fatty acids isolated from sardines in July were similar to those from plankton in the same season. This reflects the deposition of plankton fatty acids in sardine depot fat. The season of high free histidine content in the ordinary meat corresponded with that of high lipid content. These results suggested that both the fatty acid composition of sardines and the high concentrations of certain amino acids in free form are influenced by the intake and seasonal variation of composition of plankton.     

A Flower-Inducing Substance Derived from Norepinephrine upon Contact with Intact Lemna Plants

A norepinephrine solution in which intact plants of Lemna paucicostatahad been immersed for 30 min or on which intact Lemna plantshad been placed for 24 h had strong flower-inducing activityin L. paucicostata 151, but norepinephrine added to the distilledwater in which Lemna plants had been immersed had no activity. (Received May 24, 1991; Accepted July 5, 1991)     

Precise discrimination among homologous sequences by DNA probe column chromatography.

Synthesized oligo probes were immobilized to a HPLC gel in high concentrations (30-40 O.D. per gram dry gel), packed in a column (2mm x 10cm) and incorporated into a conventional HPLC system. The system was applied to the discrimination among homologous sequences. Two probes different in sequence and length (16mer and 24mer) were investigated under isothermal and isocratic conditions. For each probe, 4 oligos of similar sequences, one perfectly matched to the probe and others containing one mismatch in the center of the chain, were synthesized. The chromatogram obtained as the results of high performance liquid affinity chromatography (HPLAC) considerably varied with the column temperature and the type of mismatches. The dependency of the deformation of elution profile upon mismatch seemed to reflect the stability of the hybrid composed of samples and immobilized probe.