Single-stranded DNA used as an efficient new vehicle for transformation of plant protoplasts

In relation to the question which DNA form (single- or double-stranded) is transferred by Agrobacterium tumefaciens to plant cells, we studied the behaviour of single-stranded DNA, as compared to double-stranded DNA, when it is introduced into plant protoplasts by electroporation. To this end, we cloned a construct with a plant NPTII gene as well as a CAT gene in the M13 vectors tg130 and tg131. We found that both complementary single-stranded molecules gave rise to substantial CAT activity in plant protoplasts, suggesting that single-stranded DNA is converted into double-stranded DNA by the plant cell replication machinery. Unexpectedly, we found that single-stranded DNA leads to a 3–10 fold higher frequency of stable transformation (selection for kanamycin resistance) than double-stranded DNA. These results indicate that the use of single-stranded DNA might be considered in experiments in which optimal transformation frequencies are needed, e.g. with protoplasts form recalcitrant plant species.Abbreviations ss single-stranded - ds double-stranded - CAT chloramphenicol acetyl transferase - NPTII neomycin phosphotransferase II - RT room temperature     

Propelling efficiency of front-crawl swimming

In this study the propelling efficiency (ep) of front-crawl swimming, by use of the arms only, was calculated in four subjects. This is the ratio of the power used to overcome drag (Pd) to the total mechanical power (Po) produced including power wasted in changing the kinetic energy of masses of water (Pk). By the use of an extended version of the system to measure active drag (MAD system), Pd was measured directly. Simultaneous measurement of O2 uptake (VO2) enabled the establishment of the relationship between the rate of the energy expenditure (PVO2) and Po (since when swimming on the MAD system Po = Pd). These individual relationships describing the mechanical efficiency (8-12%) were then used to estimate Po in free swimming from measurements of VO2. Because Pd was directly measured at each velocity studied by use of the MAD system, ep could be calculated according to the equation ep = Pd/(Pd + Pk) = Pd/Po. For the four top class swimmers studied, ep was found to range from 46 to 77%. Total efficiency, defined as the product of mechanical and propelling efficiency, ranged from 5 to 8%.     

Cytoplasmic filaments and cellular wound healing in amoeba proteus

The flexibility and self-healing properties of animal cell surface membranes are well known. These properties have been best exploited in various micrurgical studies on living cells (2, 3), especially in amoebae (7, 20). During nuclear transplantation in amoebae, the hole in the membrane through which a nucleus passes can have a diameter of 20-30 μm, and yet such holes are quickly sealed, although some cytoplasm usually escapes during the transfer. While enucleating amoebae in previous studies, we found that if a very small portion of a nucleus was pushed through the membrane and exposed to the external medium, the amoeba expelled such a nucleus on its own accord. When this happened, a new membrane appeared to form around the embedded portion of the nucleus and no visible loss of cytoplasm occurred during nuclear extrusion. In the present study, we examined amoebae that were at different stages of expelling partially exposed nuclei, to follow the sequence of events during the apparent new membrane formation. Unexpectedly, we found that a new membrane is not formed around the nucleus from inside but a hole is sealed primarily by a constriction of the existing membrane, and that cytoplasmic filaments are responsible for the prevention of the loss of cytoplasm.     

One mutation,three phenotypes: novel metabolic insights on MELAS,MIDD and myopathy caused by the m.3243A > G mutation

Metabolomics - Studies investigating crop resistance to abiotic and biotic stress have largely focused on plant responses to singular forms of stress and individual biochemical pathways that only...     

Saikosaponin-d,a novel SERCA inhibitor,induces autophagic cell death in apoptosis-defective cells

Autophagy is an important cellular process that controls cells in a normal homeostatic state by recycling nutrients to maintain cellular energy levels for cell survival via the turnover of proteins and damaged organelles. However, persistent activation of autophagy can lead to excessive depletion of cellular organelles and essential proteins, leading to caspase-independent autophagic cell death. As such, inducing cell death through this autophagic mechanism could be an alternative approach to the treatment of cancers. Recently, we have identified a novel autophagic inducer, saikosaponin-d (Ssd), from a medicinal plant that induces autophagy in various types of cancer cells through the formation of autophagosomes as measured by GFP-LC3 puncta formation. By computational virtual docking analysis, biochemical assays and advanced live-cell imaging techniques, Ssd was shown to increase cytosolic calcium level via direct inhibition of sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase pump, leading to autophagy induction through the activation of the Ca²⁺/calmodulin-dependent kinase kinase–AMP-activated protein kinase–mammalian target of rapamycin pathway. In addition, Ssd treatment causes the disruption of calcium homeostasis, which induces endoplasmic reticulum stress as well as the unfolded protein responses pathway. Ssd also proved to be a potent cytotoxic agent in apoptosis-defective or apoptosis-resistant mouse embryonic fibroblast cells, which either lack caspases 3, 7 or 8 or had the Bax-Bak double knockout. These results provide a detailed understanding of the mechanism of action of Ssd, as a novel autophagic inducer, which has the potential of being developed into an anti-cancer agent for targeting apoptosis-resistant cancer cells.     

Familial hypercholesterolemia in children

PURPOSE OF THIS REVIEW: This review provides an update on recent advances in the diagnosis and management of children with familial hypercholesterolemia. RECENT FINDINGS: A large cross-sectional cohort study of paediatric familial hypercholesterolemia demonstrated that affected children had a 5-fold more rapid increase of carotid arterial wall intima-media thickness during childhood years than their affected siblings. This faster progression led to a significant deviation in terms of intima-media thickness from the age of 12 years and onwards. Low-density lipoprotein cholesterol was a strong and independent predictor of carotid artery intima-media thickness in these children, which confirms the pivotal role of low-density lipoprotein cholesterol for the development of atherosclerosis. In this condition lipid lowering by statin therapy is accompanied by carotid intima-media thickness regression in familial-hypercholesterolemic children, which suggests that initiation of low-density lipoprotein cholesterol-reducing medication in childhood already can inhibit or possibly reduce the faster progression of atherosclerosis. Furthermore, these trials demonstrated that statins are safe and do not impair growth or sexual development in these children. Conversely, products containing plant sterols reduced low-density lipoprotein cholesterol levels by 14%, but did not improve endothelial dysfunction as assessed by flow-mediated dilatation. SUMMARY: Children with familial hypercholesterolemia clearly benefit from lipid-lowering strategies. Statins are safe agents and have been proven to reduce elevated low-density lipoprotein cholesterol levels significantly. In addition, statins improve surrogate markers for atherosclerosis. Therefore these agents should become the pivotal therapy in children with familial hypercholesterolemia.     

Biochemical examination of fibroblasts in the diagnosis and research of oxidative phosphorylation (OXPHOS) defects

The oxidative phosphorylation system (OXPHOS) is organized in five multi-protein complexes, comprising four complexes (I-IV) of the respiratory chain and ATP synthase (complex V). OXPHOS has a vital role in cellular energy metabolism and ATP production. Enzyme analysis of individual OXPHOS complexes in a skeletal muscle biopsy remains the mainstay of the diagnostic process for patients suspected of mitochondrial cytopathy. A fresh muscle biopsy is preferable to a frozen muscle biopsy because of the possibility to measure the overall capacity of the OXPHOS system. In about 25% of patients referred to our center for muscle biopsy, reduced substrate oxidation rates and ATP + creatine phosphate production rates were found without any defect in complex I-V and the pyruvate dehydrogenase complex. In a subset of patients it is necessary to investigate fibroblasts for diagnostic purposes. The indications for biochemical investigations in fibroblasts are: (a) If no muscle sample is available; (b) If prenatal diagnosis is required; (c) To clarify the results obtained in muscle tissue if no clear-cut diagnosis can be made; (d) If molecular-genetic investigations are required; (e) For research purposes. Fibroblasts are less suitable than fresh muscle for investigating respiratory chain disorders, for the following reasons: (i) A defect that is present in a muscle is not always expressed in fibroblasts. (ii) Exclusion of a defect in fibroblasts does not exclude the diagnosis with regard to muscle. (iii) A specific pattern of abnormalities demonstrated in fibroblasts may not be reflected in muscle tissue. (iv) Enzyme deficiencies found in muscle are generally more pronounced than in fibroblasts. An exact diagnosis of respiratory chain defects is a prerequisite for rational therapy and genetic counseling. Provided guidelines for specimen collection are followed, there are now reliable methods for identifying respiratory chain defects.     

Insect lipoprotein biogenesis depends on an amphipathic beta cluster in apolipophorin II/I and is stimulated by microsomal triglyceride transfer protein

Lipoproteins transport lipids in the circulation of an evolutionally wide diversity of animals. The pathway for lipoprotein biogenesis has been revealed to a large extent in mammals only, in which apolipoprotein B (apoB) acquires lipids via the assistance of microsomal triglyceride transfer protein (MTP) and binds them by means of amphipathic protein structures. To investigate whether this is a common mechanism for lipoprotein biogenesis in animals, we studied the structural elements involved in the assembly of the insect lipoprotein, lipophorin. LOCATE sequence analysis predicted that the insect lipoprotein precursor, apolipophorin II/I (apoLp-II/I), contains clusters of amphipathic alpha-helices and beta-strands, organized along the protein as N-alpha(1)-beta-alpha(2)-C, reminiscent of a truncated form of apoB. Recombinant expression of a series of C-terminal truncation variants of Locusta migratoria apoLp-II/I in an insect cell (Sf9) expression system revealed that the formation of a buoyant high density lipoprotein requires the amphipathic beta cluster. Coexpression of apoLp-II/I with the MTP homolog of Drosophila melanogaster affected insect lipoprotein biogenesis quantitatively as well as qualitatively, as the secretion of apoLp-II/I proteins was increased several-fold and the buoyant density of the secreted lipoprotein decreased concomitantly, indicative of augmented lipidation. Based on these findings, we propose that, despite specific modifications, the assembly of lipoproteins involves MTP as well as amphipathic structures in the apolipoprotein carrier, both in mammals and insects. Thus, lipoprotein biogenesis in animals appears to rely on structural elements that are of early metazoan origin.