A new thermolabile alkaline phospholipase D from <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CS628

A phospholipase D (PLD₆₂₈), constitutively secreted by Streptomyces sp. CS628, was purified by ion exchange with CM Trisacryl and gel filtration with Sepharose CL-6B. The enzyme production was highest with peptone and starch as nitrogen and carbon sources, and at 30°C with an initial medium pH of 7.5. Molecular weight, optimum pH, optimum temperature, pH stability, and thermostability of the enzyme were 50 kDa, pH 9.6, 30°C, pH 5.7 ∼ 10.6 and ≤30°C, respectively. Detergents and metal ions had varied effects on the enzyme activity. Importantly, PLD₆₂₈ could not catalyze transphosphatidylation of glycerol, L-serine, myo-inositol or ethanolamine, which are extensively used to assess the activity, suggesting that PLD₆₂₈ lacks the transphosphatidylation activity. PLD₆₂₈ could be a novel PLD based on its biochemical characteristics, which are significantly different from previously reported PLDs, such as thermolability, highest activity at alkaline pH, and lack of transphosphatidylation activity.     

Diversity of cold-active protease-producing bacteria from arctic terrestrial and marine environments revealed by enrichment culture

A new approach for enrichment culture was applied to obtain cold-active protease-producing bacteria for marine and terrestrial samples from Svalbard, Norway. The method was developed for the enrichment of bacteria by long-term incubation at low temperatures in semi-solid agar medium containing meat pieces as the main source of carbon and energy. ZoBell and 0.1× nutrient broth were added for marine and terrestrial microorganisms, respectively, to supply basal elements for growth. One to three types of colonies were observed from each enrichment culture, indicating that specific bacterial species were enriched during the experimental conditions. Among 89 bacterial isolates, protease activity was observed from 48 isolates in the screening media containing skim milk. Good growth was observed at 4°C and 10°C while none of the isolates could grow at 37°C. At low temperatures, enzyme activity was equal to or higher than activity at higher temperatures. Bacterial isolates were included in the genera Pseudoalteromonas (33 isolates), Arthrobacter (24 isolates), Pseudomonas (16 isolates), Psychrobacter (6 isolates), Sphingobacterium (6 isolates), Flavobacterium (2 isolates), Sporosarcina (1 isolate), and Stenotrophomonas (1 isolate). Protease activity was observed from Pseudoalteromonas (33 isolates), Pseudomonas (10 isolates), Arthrobacter (4 isolates), and Flavobacterium (1 isolate).     

Acupuncture for functional recovery after stroke: a systematic review of sham-controlled randomized clinical trials

Background

Acupuncture is frequently advocated as an adjunct treatment during stroke rehabilitation. The aim of this review was to assess its effectiveness in this setting.

Methods

We searched 25 databases and 12 major Korean traditional medicine journals from their inception to October 2009. We included randomized controlled trials, with no language restrictions, that compared the effects of acupuncture (with or without electrical stimulation) with sham acupuncture. We assessed the methodologic quality of the trials using the Cochrane risk-of-bias criteria and the PEDro (Physiotherapy Evidence Database) scale.

Results

Ten of 664 potentially relevant studies met our inclusion criteria. For acute and subacute stages after stroke, we included seven trials. A meta-analysis of the five studies that assessed functionality did not show a significant difference in favour of acupuncture, with high heterogeneity. A post-hoc sensitivity analysis of three trials with low risk of bias did not show beneficial effects of acupuncture on activities of daily living at the end of the intervention period (n = 244; standard mean difference 0.07, 95% confidence interval [CI] −0.18 to 0.32; I² = 0%) or after follow-up (n = 244; standard mean difference 0.10, 95% CI −0.15 to 0.35; I² = 0%). For the chronic stage after stroke, three trials tested effects of acupuncture on function according to the Modified Ashworth Scale; all failed to show favourable effects.

Interpretation

Our meta-analyses of data from rigorous randomized sham-controlled trials did not show a positive effect of acupuncture as a treatment for functional recovery after stroke.Acupuncture is often used as an adjunct to mainstream rehabilitation after stroke. It involves the insertion of an acupuncture needle into the skin at certain points of the body. Acupuncture is claimed to be effective for a wide range of conditions, such as pain, musculoskeletal disorders and several neurologic diseases.¹ Possible mechanisms of its effects on neurologic conditions include stimulation of neuronal cell proliferation,² facilitation of neural plasticity,³ reduction of the post-ischemic inflammatory reaction⁴ and prevention of neuronal apoptosis.⁵Before acupuncture can be recommended for routine use, we require evidence from rigorous randomized clinical trials. In acupuncture trials, it is difficult to allow blinding of the treatment allocation.⁶ A placebo must be indistinguishable from the real treatment and inert.⁶ “Sham” is used to describe any control procedure that is used to blind treatment allocation in clinical trials of acupuncture.⁶ Several sham procedures are now available, such as the use of penetrating acupuncture on nonacupuncture points, superficial puncture of the skin on acupuncture points and nonpenetration on acupuncture points with sham needle devices.⁶Several reviews assessing the effects of acupuncture for stroke have been published. However, some did not include all of the relevant articles published in Asian countries; others included interventions other than acupuncture; and several were not systematic. We conducted a systematic review to critically evaluate all of the currently available randomized sham-controlled trials of acupuncture as an adjunct to mainstream stroke rehabilitation.     

Superoxide anion and proteasomal dysfunction contribute to curcumin-induced paraptosis of malignant breast cancer cells

Curcumin is considered a pharmacologically safe agent that may be useful in cancer chemoprevention and therapy. Here, we show for the first time that curcumin effectively induces paraptosis in malignant breast cancer cell lines, including MDA-MB-435S, MDA-MB-231, and Hs578T cells, by promoting vacuolation that results from swelling and fusion of mitochondria and/or the endoplasmic reticulum (ER). Inhibition of protein synthesis by cycloheximide blocked curcumin-induced vacuolation and subsequent cell death, indicating that protein synthesis is required for this process. The levels of AIP-1/Alix protein, a known inhibitor protein of paraptosis, were progressively downregulated in curcumin-treated malignant breast cancer cells, and AIP-1/Alix overexpression attenuated curcumin-induced death in these cells. ERK2 and JNK activation were positively associated with curcumin-induced cell death. Mitochondrial superoxide was shown to act as a critical early signal in curcumin-induced paraptosis, whereas proteasomal dysfunction was mainly responsible for the paraptotic changes associated with ER dilation. Notably, curcumin-induced paraptotic events were not observed in normal breast cells, including mammary epithelial cells and MCF-10A cells. Taken together, our findings on curcumin-induced paraptosis may provide novel insights into the mechanisms underlying the selective anti-cancer effects of curcumin against malignant cancer cells.     

Differences in accumulation of anthracyclines daunorubicin, doxorubicin and epirubicin in rat tissues revealed by immunocytochemistry

Although anthracycline antibiotics daunorubicin (DR), doxorubicin (DX), and epirubicin (ER) possess minor differences in their chemical structures, large differences are noted in their clinical use, as well as in cellular and plasma pharmacokinetic parameters in vivo. Immunocytochemistry for DR, DX, or ER was developed using an anti-DR monoclonal antibody (ADM-1-11), which has been demonstrated to react equally well with each of the three drugs, and therefore it was used for comparing their accumulation in several rat tissue cells after a single i.v. injection of each drug. In the kidney, immunoreactivity for each drug was distributed in essentially the same pattern and in the same strength 2 h after injection, but quite differently distributed in kidney cells thereafter, so that at 120-h post-injection significant amounts of DX and ER remained, but DR had almost completely vanished. Similar patterns of accumulation were observed in cells of other tissues including the pancreas, hair follicle, and stomach, with the exception of the intestine in which none of the three drugs remained after 120 h. These results appear to be supported by previous pharmacokinetic studies on the anthracyclines. The mechanism for such differences among the three drugs remains obscure, but the hydroxyl group at C-14 of DX and ER molecule might be related to the strong propensity of DX and ER to accumulate in tissue cells. The present results should contribute to the understanding of the mechanisms of the differences in the pharmacokinetics, as well as the possibly in anti-tumor activities of the anthracyclines.     

A bacterial elongation factor G homologue exclusively functions in ribosome recycling in the spirochaete Borrelia burgdorferi

Translation elongation factor G (EF‐G) in bacteria plays two distinct roles in different phases of the translation system. EF‐G catalyses the translocation of tRNAs on the ribosome in the elongation step, as well as the dissociation of the post‐termination state ribosome into two subunits in the recycling step. In contrast to this conventional view, it has very recently been demonstrated that the dual functions of bacterial EF‐G are distributed over two different EF‐G paralogues in human mitochondria. In the present study, we show that the same division of roles of EF‐G is also found in bacteria. Two EF‐G paralogues are found in the spirochaete Borrelia burgdorferi, EF‐G1 and EF‐G2. We demonstrate that EF‐G1 is a translocase, while EF‐G2 is an exclusive recycling factor. We further demonstrate that B. burgdorferi EF‐G2 does not require GTP hydrolysis for ribosome disassembly, provided that translation initiation factor 3 (IF‐3) is present in the reaction. These results indicate that two B. burgdorferi EF‐G paralogues are close relatives to mitochondrial EF‐G paralogues rather than the conventional bacterial EF‐G, in both their phylogenetic and biochemical features.     

Culture and in vitro hepatogenic differentiation of placenta-derived stem cells, using placental extract as an alternative to serum

Objectives: Translational research using adult stem cells derived from various tissues has been highlighted in cell‐based therapy. However, there are many limitations to using conventional culture systems of adult stem cells for clinically applicability, including limited combinations of cytokines and use of nutrients derived from animals. Here, we have investigated the effects of placental extract (PE) for culture of placenta‐derived stem cells (PDSCs) as well as their potential for hepatogenic differentiation. Materials and methods: Placental extract, extracted using water‐soluble methods, was used as a supplement for culture of PDSCs. Cell viability was determined using the MTT assay, and cytokine assay was performed using Luminex assay kit. Gene expression, indocyanine green (ICG) up‐take, PAS (Periodic Acid‐Schiff) staining and urea production were also analysed. Results: The placental extract contained several types of cytokine and chemokine essential for maintenance and differentiation of stem cells. Expression of stemness markers in PDSCs cultured with PE is no different from that of PDSCs cultured with foetal bovine serum (FBS). After hepatogenic differentiation, expression patterns for hepatocyte‐specific markers in PDSCs cultured with PE were consistent and potential for hepatogenic differentiation of PDSCs cultured with PE was similar to that of PDSCs cultured with FBS, as shown by PAS staining and urea production assays. Conclusions: Our findings revealed that placental extract could be used as a new component for culture of adult stem cells, as well as for development of human‐based medium, in translational research for regenerative medicine.     

Seasonal changes of functional groups in coleopteran communities in pine forests

Fauna assemblages reflect their habitat relating to ecological function in an ecosystem. The functional groups are concerned with how a resource is processed by different species to provide a specific ecosystem service or function. We elucidated seasonal changes of coleopteran functional groups in forests, and evaluated their ecological roles related to available food resources. Coleopteran communities were collected weekly or biweekly using Malaise traps at nine study sites in Japanese red pine forests in Korea from late June to September 2005. Compositions of the functional groups were compared at the different sites and at sampling times with respect to taxa richness and abundance. Cluster analysis and non-metric multidimensional scaling were used to characterize spatial and temporal changes of functional groups. Herbivores and dead/live wood feeders regulating primary production in the pine forests were the dominant coleopteran groups in July, followed by detritivores and predators that dominated from July to August, resulting from the accumulation of detritus. Then, fungivores became dominant due to increased fungal biomass in the forest. Seasonal changes of coleopteran functional groups shifted from regulators of primary production to regulators of decomposition, reflecting their available food resources. In addition, abundance of detritivores and predators were dependent on total abundance of coleopterans, suggesting that these two groups reflect their habitat condition.