Isolation and culture of protoplasts of pigeonpea (Cajanus cajan L.)

Summary High yields of protoplasts were obtained from leaves of aseptically grown plants and calli originated from different explants, in several cultivars of Cajanus cajan L. The protoplasts divided to form cell clusters in modified KM 8p medium and developed to protocolonies after dilution with liquid Caboche's medium within three to four weeks of culture. The protocolonies proliferated to form green calli on solid Caboche's medium. No shoots or plants were obtained.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kin kinetin - Zea zeatin - Adn S adenine sulphate - GA ₃ gibberellic acid     

Artificial mutations and natural variations in the CD46 molecules from human and monkey cells define regions important for measles virus binding.

CD46 was previously shown to be a primate-specific receptor for the Edmonston strain of measles virus. This receptor consists of four short consensus regions (SCR1 to SCR4) which normally function in complement regulation. Measles virus has recently been shown to interact with SCR1 and SCR2. In this study, receptors on different types of monkey erythrocytes were employed as "natural mutant proteins" to further define the virus binding regions of CD46. Erythrocytes from African green monkeys and rhesus macaques hemagglutinate in the presence of measles virus, while baboon erythrocytes were the least efficient of the Old World monkey cells used in these assays. Subsequent studies demonstrated that the SCR2 domain of baboon CD46 contained an Arg-to-Gln mutation at amino acid position 103 which accounted for reduced hemagglutination activity. Surprisingly, none of the New World monkey erythrocytes hemagglutinated in the presence of virus. Sequencing of cDNAs derived from the lymphocytes of these New World monkeys and analysis of their erythrocytes with SCR1-specific polyclonal antibodies indicated that the SCR1 domain was deleted in these cells. Additional experiments, which used 35 different site-specific mutations inserted into CD46, were performed to complement the preceding studies. The effects of these artificial mutations were documented with a convenient binding assay using insect cells expressing the measles virus hemagglutinin. Mutations which mimicked the change found in baboon CD46 or another which deleted the SCR2 glycosylation site reduced binding substantially. Another mutation which altered GluArg to AlaAla at positions 58 and 59, totally abolished binding. Finally, the epitopes for two monoclonal antibodies which inhibit measles virus attachment were mapped to the same regions implicated by mutagenesis.     

Correction to: Sex and race differences of cerebrospinal fluid metabolites in healthy individuals

Metabolomics - Metabolomics applications to the aquaculture research are increasing steadily. The use of standardized proton nuclear magnetic resonance (1H NMR) spectroscopy can provide the...     

Design Maps for the Hyperthermic Treatment of Tumors with Superparamagnetic Nanoparticles

A plethora of magnetic nanoparticles has been developed and investigated under different alternating magnetic fields (AMF) for the hyperthermic treatment of malignant tissues. Yet, clinical applications of magnetic hyperthermia are sporadic, mostly due to the low energy conversion efficiency of the metallic nanoparticles and the high tissue concentrations required. Here, we study the hyperthermic performance of commercially available formulations of superparamagnetic iron oxide nanoparticles (SPIOs), with core diameter of 5, 7 and 14 nm, in terms of absolute temperature increase ΔT and specific absorption rate (SAR). These nanoparticles are operated under a broad range of AMF conditions, with frequency f varying between 0.2 and 30 MHz; field strength H ranging from 4 to 10 kA m⁻¹; and concentration c_MNP varying from 0.02 to 3.5 mg ml⁻¹. At high frequency field (∼30 MHz), non specific heating dominates and ΔT correlates with the electrical conductivity of the medium. At low frequency field (<1 MHz), non specific heating is negligible and the relaxation of the SPIO within the AMF is the sole energy source. We show that the ΔT of the medium grows linearly with c_MNP, whereas the SAR_MNP of the magnetic nanoparticles is independent of c_MNP and varies linearly with f and H². Using a computational model for heat transport in a biological tissue, the minimum requirements for local hyperthermia (T_tissue >42°C) and thermal ablation (T_tissue >50°C) are derived in terms of c_MNP, operating AMF conditions and blood perfusion. The resulting maps can be used to rationally design hyperthermic treatments and identifying the proper route of administration – systemic versus intratumor injection – depending on the magnetic and biodistribution properties of the nanoparticles.     

X protein of hepatitis B virus inhibits Fas-mediated apoptosis and is associated with up-regulation of the SAPK/JNK pathway

The X protein from a chronic strain of hepatitis B virus (HBx) was determined to inhibit Fas-mediated apoptosis and promote cell survival. Fas-mediated apoptosis is the major cause of hepatocyte damage during liver disease. Experiments demonstrated that cell death caused by anti-Fas antibodies was blocked by the expression of HBx in human primary hepatocytes and mouse embryo fibroblasts. This effect was also observed in mouse erythroleukemia cells that lacked p53, indicating that protection against Fas-mediated apoptosis was independent of p53. Components of the signal transduction pathways involved in this protection were studied. The SAPK/JNK pathway has previously been suggested to be a survival pathway for some cells undergoing Fas-mediated apoptosis, and kinase assays showed that SAPK activity was highly up-regulated in cells expressing the HBx protein. Normal mouse fibroblasts expressing HBx were protected from death, whereas identical fibroblasts lacking the SEK1 component from the SAPK pathway succumbed to Fas-mediated apoptosis, whether HBx was present or not. Assays showed that caspase 3 and 8 activities and the release of cytochrome c from mitochondria were inhibited, in the presence of HBx, following stimulation with anti-Fas antibodies. Coprecipitation and confocal immunofluorescence microscopy experiments demonstrated that HBx localizes with a cytoplasmic complex containing MEKK1, SEK1, SAPK, and 14-3-3 proteins. Finally, mutational analysis of HBx demonstrated that a potential binding region for 14-3-3 proteins was essential for induction of SAPK/JNK activity and protection from Fas-mediated apoptosis.     

Sphingomyelin metabolites inhibit sphingomyelin synthase and CTP:phosphocholine cytidylyltransferase

Tissue injury in inflammation involves the release of several cytokines that activate sphingomyelinases and generate ceramide. In the lung, the impaired metabolism of surfactant phosphatidylcholine (PC) accompanies this acute and chronic injury. These effects are long-lived and extend beyond the time frame over which tumor necrosis factor (TNF)-alpha and interleukin-1beta are elevated. In this paper, we demonstrate that in H441 lung cells these two processes, cytokine-induced metabolism of sphingomyelin and the inhibition of PC metabolism, are directly interrelated. First, metabolites of sphingomyelin hydrolysis themselves inhibit key enzymes necessary for restoring homeostasis between sphingomyelin and its metabolites. Ceramide stimulates sphingomyelinases as effectively as TNF-alpha, thereby amplifying the sphingomyelinase activation, and TNF-alpha, ceramide, and sphingosine all inhibit PC:ceramide phosphocholine transferase (sphingomyelin synthase), the enzyme that restores homeostasis between sphingomyelin and ceramide pools. Second, ceramide inhibits PC synthesis, probably because of its effects on CTP:phosphocholine cytidylyltransferase, the rate-limiting enzymatic step in de novo PC synthesis. The data presented here suggest that TNF-alpha may be an inhibitor of phospholipid metabolism in inflammatory tissue injury. These actions may be amplified because of the ability of metabolites of sphingomyelin to inhibit the pathways that should restore the normal ceramide-sphingomyelin homeostasis.     

Modified apoptotic molecule (BID) reduces hepatitis C virus infection in mice with chimeric human livers

Hepatitis C virus (HCV) encodes a polyprotein consisting of core, envelope (E1, E2, p7), and nonstructural polypeptides (NS2, NS3, NS4A, NS4B, NS5A, NS5B). The serine protease (NS3/NS4A), helicase (NS3), and polymerase (NS5B) constitute valid targets for antiviral therapy. We engineered BH3 interacting domain death agonist (BID), an apoptosis-inducing molecule, to contain a specific cleavage site recognized by the NS3/NS4A protease. Cleavage of the BID precursor molecule by the viral protease activated downstream apoptotic molecules of the mitochondrial pathway and triggered cell death. We extended this concept to cells transfected with an infectious HCV genome, hepatocytes containing HCV replicons, a Sindbis virus model for HCV, and finally HCV-infected mice with chimeric human livers. Infected mice injected with an adenovirus vector expressing modified BID exhibited HCV-dependent apoptosis in the human liver xenograft and considerable declines in serum HCV titers.