Effect of cobalt-60 (γ radiation) on multidrug-resistant multiple myeloma cell lines

Emergence of resistance to chemotherapy and radiotherapy is a major obstacle for the successful treatment of MM (multiple myeloma). Prednisone, vincristine and melphalan are commonly used chemotherapeutic agents for the treatment of MM. In the current study, we examined the presence of possible cross-resistance between these drugs and gamma (γ) radiation. Prednisone, vincristine and melphalan resistant RPMI-8226 and U-266 MM cells were generated by stepwise increasing concentrations of the drugs. The sensitive and resistant cells were exposed to 200- and 800 cGy γ radiation, and proliferation was examined by XTT {2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide} assay. The results showed that Prednisone- and melphalan-resistant RPMI-8226 cells were also cross-resistant to 200 and 800 cGy γ radiation application, while vincristine-resistant cells did not show resistance. On the other hand, Prednisone-, vincristine- and melphalan-resistant U-266 cells showed cross-resistance to 200- and 800 cGy γ radiation application. These results demonstrated that MM cells resistant to anticancer agents respond to radiation in different levels. These findings may be important in the clinical applications of radiation therapy in the treatment of vincristine resistant MM.     

Comparison of the physiological responses to different small-sided games in elite young soccer players

The purpose of this study was to compare the blood lactate (La-), heart rate (HR) and percentage of maximum HR (%HRmax) responses among the small-sided games (SSGs) in elite young soccer players. Sixteen players (average age 15.7 6 0.4 years; height 176.8 6 4.6 cm; body mass 65.5 6 5.6 kg; VO2max 53.1 6 5.9 ml · kg(-1) · min(-1); HRmax 195.9 6 7.4 b · min(-1)) volunteered to perform the YoYo intermittent recovery test and 6 bouts of soccer drills including 1-a-side, 2-a-side, 3-a-side, and 4-a-side games without a goalkeeper in random order at 2-day intervals. The differences in La-, HR and%HRmax either among the SSGs or among the bouts were identified using 4 x 6 (games x exercise bouts) 2-way analysis of variance with repeated measures. Significant differences were found on La-, HR, and %HRmax among the bouts (p ≤ 0.05). The 3-a-side and 4-a-side games were significantly higher than 1-a-side and 2-a-side games on HR and %HRmax (p ≤ 0.05), whereas the 1-a-side game significantly resulted in higher La- responses compared to other SSGs. This study demonstrated that physiological responses during the 1-a-side and 2-a-side games were different compared to 3-a-side and 4-a-side games. Therefore, it can be concluded that a decreased number of players results in increased intensity during SSGs including 6 bouts. These results suggest that coaches should pay attention on choosing the SSG type and the number of bouts to improve desired physical conditioning of elite young soccer players in soccer training.     

Signposts ahead: Hard tissue signals on rue Armand de Ricqlès

Of the major contributions to our understanding of the skeleton made by Armand de Ricqlès is the notion that within the microanatomy of bone we may observe “signals”, some relating to phylogeny, and others to aspects of growth, function, and physiology. We are motivated to follow this road, as it were, and read the “signposts” along the way. Incremental structures are such signposts, representing biological rhythms as successive forming fronts in enamel and bone. A long period rhythm in humans, which occurs on average every eight to nine days, is observed in enamel as the stria of Retzius and in bone as the lamella. Because lamellae are formed within defined periods of time, quantitative measures of widths of individual lamellae provide time-resolved growth rate variability. Results obtained on primary incremental lamellar bone from midshaft femur histological sections of sub-Saharan Africans of Bantu origin and known life history reveal environmental effects heretofore unknown.     

Improved malaria case management through the implementation of a health facility-based sentinel site surveillance system in Uganda

Background

Heath facility-based sentinel site surveillance has been proposed as a means of monitoring trends in malaria morbidity but may also provide an opportunity to improve malaria case management. Here we described the impact of a sentinel site malaria surveillance system on promoting laboratory testing and rational antimalarial drug use.

Methodology/Principal Findings

Sentinel site malaria surveillance was established at six health facilities in Uganda between September 2006 and January 2007. Data were collected from all patients presenting to the outpatient departments including demographics, laboratory results, diagnoses, and treatments prescribed. Between the start of surveillance and March 2010, a total 424,701 patients were seen of which 229,375 (54%) were suspected of having malaria. Comparing the first three months with the last three months of surveillance, the proportion of patients with suspected malaria who underwent diagnostic testing increased from 39% to 97% (p<0.001). The proportion of patients with an appropriate decision to prescribe antimalarial therapy (positive test result prescribed, negative test result not prescribed) increased from 64% to 95% (p<0.001). The proportion of patients appropriately prescribed antimalarial therapy who were prescribed the recommended first-line regimen artemether-lumefantrine increased from 48% to 69% (p<0.001).

Conclusions/Significance

The establishment of a sentinel site malaria surveillance system in Uganda achieved almost universal utilization of diagnostic testing in patients with suspected malaria and appropriate decisions to prescribed antimalarial based on test results. Less success was achieved in promoting prescribing practice for the recommended first-line therapy. This system could provide a model for improving malaria case management in other health facilities in Africa.     

Involvement of Acid ��-Glucosidase 1 in the Salvage Pathway of Ceramide Formation

Activation of protein kinase C (PKC) promotes the salvage pathway of ceramide formation, and acid sphingomyelinase has been implicated, in part, in providing substrate for this pathway (Zeidan, Y. H., and Hannun, Y. A. (2007) J. Biol. Chem. 282, 11549–11561). In the present study, we examined whether acid β-glucosidase 1 (GBA1), which hydrolyzes glucosylceramide to form lysosomal ceramide, was involved in PKC-regulated formation of ceramide from recycled sphingosine. Glucosylceramide levels declined after treatment of MCF-7 cells with a potent PKC activator, phorbol 12-myristate 13-acetate (PMA). Silencing GBA1 by small interfering RNAs significantly attenuated acid glucocerebrosidase activity and decreased PMA-induced formation of ceramide by 50%. Silencing GBA1 blocked PMA-induced degradation of glucosylceramide and generation of sphingosine, the source for ceramide biosynthesis. Reciprocally, forced expression of GBA1 increased ceramide levels. These observations indicate that GBA1 activation can generate the source (sphingosine) for PMA-induced formation of ceramide through the salvage pathway. Next, the role of PKCδ, a direct effector of PMA, in the formation of ceramide was determined. By attenuating expression of PKCδ, cells failed to trigger PMA-induced alterations in levels of ceramide, sphingomyelin, and glucosylceramide. Thus, PKCδ activation is suggested to stimulate the degradation of both sphingomyelin and glucosylceramide leading to the salvage pathway of ceramide formation. Collectively, GBA1 is identified as a novel source of regulated formation of ceramide, and PKCδ is an upstream regulator of this pathway.Sphingolipids are abundant components of cellular membranes, many of which are emerging as bioactive lipid mediators thought to play crucial roles in cellular responses (1, 2). Ceramide, a central sphingolipid, serves as the main precursor for various sphingolipids, including glycosphingolipids, gangliosides, and sphingomyelin. Regulation of formation of ceramide has been demonstrated through the action of three major pathways: the de novo pathway (3, 4), the sphingomyelinase pathway (5), and the salvage pathway (6–8). The latter plays an important role in constitutive sphingolipid turnover by salvaging long-chain sphingoid bases (sphingosine and dihydrosphingosine) that serve as sphingolipid backbones for ceramide and dihydroceramide as well as all complex sphingolipids (Fig. 1A).Open in a separate windowFIGURE 1.The scheme of the sphingosine salvage pathway of ceramide formation and inhibition of PMA induction of ceramide by fumonisin B1. A, the scheme of the sphingosine salvage pathway of ceramide formation. B, previously published data as to effects of fumonisin B1 on ceramide mass profiles (23) are re-plotted as a PMA induction of ceramide. In brief, MCF-7 cells were pretreated with or without 100 μm fumonisin B1 for 2 h followed by treatment with 100 nm PMA for 1 h. Lipids were extracted, and then the levels of ceramide species were determined by high-performance liquid chromatography-tandem mass spectrometry. Results are expressed as sum of increased mass of ceramide species. Dotted or open columns represents C₁₆-ceramide or sum of other ceramide species (C₁₄-ceramide, C₁₈-ceramide, C_18:1-ceramide, C₂₀-ceramide, C₂₄-ceramide, and C_24:1-ceramide), respectively. The data represent mean ± S.E. of three to five values.Metabolically, ceramide is also formed from degradation of glycosphingolipids (Fig. 1A) usually in acidic compartments, the lysosomes and/or late endosomes (9). The stepwise hydrolysis of complex glycosphingolipids eventually results in the formation of glucosylceramide, which in turn is converted to ceramide by the action of acid β-glucosidase 1 (GBA1)² (9, 10). Severe defects in GBA1 activity cause Gaucher disease, which is associated with aberrant accumulation of the lipid substrates (10–14). On the other hand, sphingomyelin is cleaved by acid sphingomyelinase to also form ceramide (15, 16). Either process results in the generation of lysosomal ceramide that can then be deacylated by acid ceramidase (17), releasing sphingosine that may escape the lysosome (18). The released sphingosine may become a substrate for either sphingosine kinases or ceramide synthases, forming sphingosine 1-phosphate or ceramide, respectively (3, 19–21).In a related line of investigation, our studies (20, 22, 23) have begun to implicate protein kinase Cs (PKC) as upstream regulators of the sphingoid base salvage pathway resulting in ceramide synthesis. Activation of PKCs by the phorbol ester (PMA) was shown to stimulate the salvage pathway resulting in increases in ceramide. All the induced ceramide was inhibited by pretreatment with a ceramide synthase inhibitor, fumonisin B1, but not by myriocin, thus negating acute activation of the de novo pathway and establishing a role for ceramide synthesis (20, 23). Moreover, labeling studies also implicated the salvage pathway because PMA induced turnover of steady state-labeled sphingolipids but did not affect de novo labeled ceramide in pulse-chase experiments.Moreover, PKCδ, among PKC isoforms, was identified as an upstream molecule for the activation of acid sphingomyelinase in the salvage pathway (22). Interestingly, the PKCδ isoform induced the phosphorylation of acid sphingomyelinase at serine 508, leading to its activation and consequent formation of ceramide. The activation of acid sphingomyelinase appeared to contribute to ∼50% of the salvage pathway-induced increase in ceramide (28) (also, see Fig. 4C). This raised the possibility that distinct routes of ceramide metabolism may account for the remainder of ceramide generation. In this study, we investigated glucocerebrosidase GBA1 as a candidate for one of the other routes accounting for PKC-regulated salvage pathway of ceramide formation.Open in a separate windowFIGURE 4.Effects of knockdown of lysosomal enzymes on the generation of ceramide after PMA treatment. A, MCF-7 cells were transfected with 5 nm siRNAs of each of four individual sequences (SCR, GBA1-a, GBA1-b, and GBA1-c) for 48 h and then stimulated with 100 nm PMA for 1 h. Lipids were extracted, and then the levels of the C₁₆-ceramide species were determined by high-performance liquid chromatography-tandem mass spectrometry. The data represent mean ± S.E. of three to nine values. B, MCF-7 cells were transfected with 5 nm siRNAs of SCR or GBA1-a (GBA1) for 48 h and then stimulated with 100 nm PMA for 1 h. Lipids were extracted, and then the levels of individual ceramide species were determined by high-performance liquid chromatography-tandem mass spectrometry. The data represent mean ± S.E. of three to five values. C₁₄-Cer, C₁₄-ceramide; C₁₆-Cer, C₁₆-ceramide; C₁₈-Cer; C₁₈-ceramide; C_18:1-Cer, C_18:1-ceramide; C₂₀-Cer, C₂₀-ceramide; C₂₀-Cer, C₂₄-ceramide; C_24:1-Cer, C_24:1-ceramide. C, MCF-7 cells were transfected with 5 nm siRNAs of SCR, acid sphingomyelinase (ASM), or GBA1-a (GBA1) for 48 h following stimulation with (PMA) or without (Control) 100 nm PMA for 1 h. Lipids were extracted, and then the levels of ceramide species were determined by high-performance liquid chromatography-tandem mass spectrometry. Levels of C₁₆-ceramide are shown. The data represent mean ± S.E. of four to five values. Significant changes from SCR-transfected cells treated with PMA are shown in A–C (^*, p < 0.02; ^**, p < 0.05; ^***, p < 0.01).