Effect of Tyrosine on the Production of Alkaloids in the High-Yielding Cell Lines of Papaver somniferum Tissue Culture

The high yielding cell lines were isolated from the heterogenous callus culture of Papaver somniferum established on modified Murashige and Skoog’s medium. These cell lines were transferred to liquid medium, and maintained for six months by frequent subculturing. The tissues were supplied with different concentrations (12.5, 25, 50 and 100 mg/100 ml) of tyrosine and analysed quantitatively for their alkaloidal contents. Six major opium alkaloids-morphine, codeine, thebaine, narceine, narcotine and papaverine, were identified. The tissue grown on liquid medium supplemented with 12.5 mg tyrosine/100 ml showed maximum percentage of alkaloids and therefore this concentration is considered as the most favourable condition and can be utilized for the large scale production of alkaloids from the cell lines.     

Identification of surfactant proteolipid SP-B in human surfactant and fetal lung

Surfactant proteolipid (SP-B) is one of several hydrophobic peptides detected in organic extracts of pulmonary surfactant and associated with the dramatic surface-active properties of surfactant phospholipids. In the present study human SP-B was identified as a protein with a relative molecular weight (Mr) of 7,500-8,000 under reducing conditions; protein of Mr 18,000 was detected under nonreducing conditions by immunoblot analysis of organic extracts of bovine and human surfactant utilizing an antiserum directed against a 60-amino acid synthetic SP-B peptide. This peptide antiserum was subsequently used to identify SP-B in explant cultures of 18- to 23-wk gestation human fetal lung. Immunoprecipitation of explants labeled with [35S]methionine after 48 h of culture identified proteins of Mr 40,000-42,000, 25,000, and 18,000 after electrophoresis under nonreducing conditions. The Mr 18,000 form was reduced to Mr 7,500-8,000 in the presence of beta-mercaptoethanol. These molecular forms likely represent the SP-B precursor protein, a proteolytic intermediate, and the mature SP-B peptide, respectively. Immunocytochemistry with the peptide antiserum localized SPL(Phe) in granular inclusions in the apical region of type II-like epithelial cells, a pattern of staining similar to that observed for the major surfactant-associated protein of Mr 26,000-38,000 (SP-A). SP-B is a novel pulmonary surfactant-associated protein that is synthesized by the human alveolar type II epithelial cell as an Mr 40,000-42,000 precursor that is subsequently proteolytically processed to Mr 7,500-8,000.     

Alterations in c-Myc phenotypes resulting from dynamin-related protein 1 (Drp1)-mediated mitochondrial fission

The c-Myc (Myc) oncoprotein regulates numerous phenotypes pertaining to cell mass, survival and metabolism. Glycolysis, oxidative phosphorylation (OXPHOS) and mitochondrial biogenesis are positively controlled by Myc, with myc−/− rat fibroblasts displaying atrophic mitochondria, structural and functional defects in electron transport chain (ETC) components, compromised OXPHOS and ATP depletion. However, while Myc influences mitochondrial structure and function, it is not clear to what extent the reverse is true. To test this, we induced a state of mitochondrial hyper-fission in rat fibroblasts by de-regulating Drp1, a dynamin-like GTPase that participates in the terminal fission process. The mitochondria from these cells showed reduced mass and interconnectivity, a paucity of cristae, a marked reduction in OXPHOS and structural and functional defects in ETC Complexes I and V. High rates of abortive mitochondrial fusion were observed, likely reflecting ongoing, but ultimately futile, attempts to normalize mitochondrial mass. Cellular consequences included reduction of cell volume, ATP depletion and activation of AMP-dependent protein kinase. In response to Myc deregulation, apoptosis was significantly impaired both in the absence and presence of serum, although this could be reversed by increasing ATP levels by pharmacologic means. The current work demonstrates that enforced mitochondrial fission closely recapitulates a state of Myc deficiency and that mitochondrial integrity and function can affect Myc-regulated cellular behaviors. The low intracellular ATP levels that are frequently seen in some tumors as a result of inadequate vascular perfusion could favor tumor survival by countering the pro-apoptotic tendencies of Myc overexpression.     

Protein glycosylation mutants of procyclic Trypanosoma brucei: defects in the asparagine-glycosylation pathway

We employed a genetic approach to study protein glycosylation in the procyclic form of the parasite Trypanosoma brucei. Two different mutant parasites, ConA 1-1 and ConA 4-1, were isolated from mutagenized cultures by selecting cells which resisted killing or agglutination by concanavalin A. Both mutant cells show reduced concanavalin A binding. However, the mutants have different phenotypes, as indicated by the fact that ConA 1-1 binds to wheat germ agglutinin but ConA 4-1 and wild type do not. A blot probed with concanavalin A revealed that many proteins in both mutants lost the ability to bind this lectin, and the blots resembled one of wild type membrane proteins treated with PNGase F. This finding suggested that the mutants had altered asparagine- linked glycosylation. This conclusion was confirmed by studies on a flagellar protein (Fla1) and procyclic acidic repetitive protein (PARP). Structural analysis indicated that the N- glycan of wild type PARP is exclusively Man5GlcNAc2 whereas that in both mutants is predominantly a hybrid type with a terminal N- acetyllactosamine. The occupancy of the PARP glycosylation site in ConA 4-1 was much lower than that in ConA 1-1. These mutants will be useful for studying trypanosome glycosylation mechanisms and function.      

A novel insecticidal GroEL protein from Xenorhabdus nematophila confers insect resistance in tobacco

Xenorhabdus nematophila is an entomopathogenic bacteria. It secretes a GroEL homolog, XnGroEL protein, toxic to its larval prey. GroEL belongs to the family of molecular chaperones and is required for proper folding of cellular proteins. Oral ingestion of insecticidal XnGroEL protein is toxic to Helicoverpa armigera, leading to cessation of growth and development of the larvae. In the present study, the insecticidal efficacy of XnGroEL against H. armigera has been evaluated in transgenic tobacco plant expressing the protein. A 1.7-kb gene encoding the 58-kDa XnGroEL protein was incorporated into the tobacco genome via Agrobacterium-mediated transformation. The stable integration of the transgene was confirmed by Southern blot analysis and its expression by RT-PCR and western blot analyses in transgenic plants. The transgenic lines showed healthy growth and were phenotypically normal. Insect bioassays revealed significant reduction of 100 % in the survival of larvae (p < 0.001) and 55–77 % reduction in plant damage (p < 0.05 and p < 0.001) compared to the untransformed and vector control plants. The results demonstrate that XnGroEL is a novel potential candidate for imparting insect resistance against H. armigera in plants.     

Changes in end-to-end interactions of tropomyosin affect mouse cardiac muscle dynamics

The ends of striated muscle tropomyosin (TM) are integral for thin filament cooperativity, determining the cooperative unit size and regulating the affinity of TM for actin. We hypothesized that altering the alpha-TM carboxy terminal overlap end to the beta-TM counterpart would affect the amino-terminal association, which would alter the end-to-end interactions of TM molecules in the thin filament regulatory strand and affect the mechanisms of cardiac muscle contraction. To test this hypothesis, we generated transgenic (TG) mouse lines that express a mutant form of alpha-TM in which the first 275 residues are from alpha-TM and the last nine amino acids are from beta-TM (alpha-TM9aaDeltabeta). Molecular analyses show that endogenous alpha-TM mRNA and protein are nearly completely replaced with alpha-TM9aaDeltabeta. Working heart preparations data show that the rates of contraction and relaxation are reduced in alpha-TM9aaDeltabeta hearts. Left ventricular pressure and time to peak pressure are also reduced (-12% and -13%, respectively). The ratio of maximum to minimum first derivatives of change in left ventricular systolic pressure with respect to time (ratio of +dP/dt to -dP/dt, respectively) is increased, but tau is not changed significantly. Force-intracellular calcium concentration ([Ca2+]i) measurements from intact papillary fibers demonstrate that alpha-TM9aaDeltabeta TG fibers produce less force per given [Ca2+]i compared with nontransgenic fibers. Taken together, the data demonstrate that the rate of contraction is primarily affected in TM TG hearts. Protein docking studies show that in the mutant molecule, the overall carbon backbone is perturbed about 1.5 A, indicating that end-to-end interactions are altered. These results demonstrate that the localized flexibility present in the coiled-coil structures of TM isoforms is different, and that plays an important role in interacting with neighboring thin filament regulatory proteins and with differentially modulating the myofilament activation processes.