Phenotypical temperature adaptation of protein synthesis in wheat seedlings: time curves for readaptation

Optimum temperature and temperature coefficient of protein synthesis in young wheat plants exhibit phenotypical temperature adaptation. In plants grown for 2 days at either chilling (4 C), medium (20 C), or high (36 C) temperature the respective values are: 27 C and 14.2 kilocalories per mole, 31 C and 18.2 kilocalories per mole, 35 C and 23.6 kilocalories per mole, based on in vivo [¹⁴C]leucine incorporation into total protein. The validity of the [¹⁴C]leucine incubation method has been confirmed by double-labeling experiments. Readaptation time curves are complex: the optimum temperature parameter readjusts within approximately 4 hours to an altered temperature regime, whereas the temperature coefficient needs between 4 and 96 hours for complete readaptation—depending on the temperature conditions prior to the temperature shift. Heat-preadapted plants need a recovery period at medium temperature to regain their cold adaptability with respect to optimum temperature. Cycloheximide (30 micrograms per milliliter) reduces [¹⁴C]leucine incorporation into protein by 85%, thus indicating that predominantly the cytoplasmic 80S system of protein synthesis is involved in temperature adaptation.     

GABAergic Signaling Is Linked to a Hypermigratory Phenotype in Dendritic Cells Infected by Toxoplasma gondii

During acute infection in human and animal hosts, the obligate intracellular protozoan Toxoplasma gondii infects a variety of cell types, including leukocytes. Poised to respond to invading pathogens, dendritic cells (DC) may also be exploited by T. gondii for spread in the infected host. Here, we report that human and mouse myeloid DC possess functional γ-aminobutyric acid (GABA) receptors and the machinery for GABA biosynthesis and secretion. Shortly after T. gondii infection (genotypes I, II and III), DC responded with enhanced GABA secretion in vitro. We demonstrate that GABA activates GABA_A receptor-mediated currents in T. gondii-infected DC, which exhibit a hypermigratory phenotype. Inhibition of GABA synthesis, transportation or GABA_A receptor blockade in T. gondii-infected DC resulted in impaired transmigration capacity, motility and chemotactic response to CCL19 in vitro. Moreover, exogenous GABA or supernatant from infected DC restored the migration of infected DC in vitro. In a mouse model of toxoplasmosis, adoptive transfer of infected DC pre-treated with GABAergic inhibitors reduced parasite dissemination and parasite loads in target organs, e.g. the central nervous system. Altogether, we provide evidence that GABAergic signaling modulates the migratory properties of DC and that T. gondii likely makes use of this pathway for dissemination. The findings unveil that GABA, the principal inhibitory neurotransmitter in the brain, has activation functions in the immune system that may be hijacked by intracellular pathogens.     

Degradation of the Met tyrosine kinase receptor by the ubiquitin-proteasome pathway.

The Met tyrosine kinase receptor is a widely expressed molecule which mediates pleiotropic cellular responses following activation by its ligand, hepatocyte growth factor/scatter factor (HGF/SF). In this communication we demonstrate that significant Met degradation is induced by HGF/SF and that this degradation can be blocked by lactacystin, an inhibitor of proteasome activity. We also show that Met is rapidly polyubiquitinated in response to ligand and that polyubiquitinated Met molecules, which are normally unstable, are stabilized by lactacystin. Both HGF/SF-induced degradation and polyubiquitination of Met were shown to be dependent on the receptor possessing intact tyrosine kinase activity. Finally, we found that a normally highly labile 55-kDa fragment of the Met receptor is stabilized by lactacystin and demonstrate that it represents a cell-associated remnant that is generated following the ligand-independent proteolytic cleavage of the Met receptor in its extracellular domain. This truncated Met molecule encompasses the kinase domain of the receptor and is itself tyrosine phosphorylated. We conclude that the ubiquitin-proteasome pathway plays a significant role in the degradation of the Met tyrosine kinase receptor as directed by ligand-dependent and -independent signals. We propose that this proteolytic pathway may be important for averting cellular transformation by desensitizing Met signaling following ligand stimulation and by eliminating potentially oncogenic fragments generated via extracellular cleavage of the Met receptor.     

Lag-phases in phytochrome-mediated enzyme synthesis (PAL)

Summary In experiments with the mustard seedling (Sinapis alba L.) it was confirmed that in the case of secondary irradiation induction of PAL by phytochrome is a very rapid process. The lag-phase after the onset of light is too brief to be detected. However, the data of other investigators, who found extended secondary lag-phases in their experimental material, can be imitated with the mustard seedling. We explain why these investigators were not able to eliminate the secondary lag-phase.PAL=Phenylalanine ammonia-lyase (EC.4.3.1.5).     

Ex-vivo models of the Retinal Pigment Epithelium (RPE) in long-term culture faithfully recapitulate key structural and physiological features of native RPE

The Retinal Pigment Epithelium (RPE) forms the primary site of pathology in several blinding retinopathies. RPE cultures are being continuously refined so that dynamic disease processes in this important monolayer can be faithfully studied outside the eye over longer periods. The RPE substrate, which mimics the supportive Bruch’s membrane (BrM), plays a key role in determining how well in-vitro cultures recapitulate native RPE cells. Here, we evaluate how two different types of BrM substrates; (1) a commercially-available polyester transwell membrane, and (2) a novel electrospun scaffold developed in our laboratory, could support the generation of realistic RPE tissues in culture. Our findings reveal that both substrates were capable of supporting long-lasting RPE monolayers with structural and functional specialisations of in-situ RPE cells. These cultures were used to study autofluorescence and barrier formation, as well as activities such as outer-segment internalisation/trafficking and directional secretion of key proteins; the impairment of which underlies retinal disease. Hence, both substrates fulfilled important criteria for generating authentic in-vitro cultures and act as powerful tools to study RPE pathophysiology. However, RPE grown on electrospun scaffolds may be better suited to studying complex RPE-BrM interactions such as the formation of drusen-like deposits associated with early retinal disease.     

Genomics insights into symbiotic nitrogen fixation

Following an interaction with rhizobial soil bacteria, legume plants are able to form a novel organ, termed the root nodule. This organ houses the rhizobial microsymbionts, which perform the biological nitrogen fixation process resulting in the incorporation of ammonia into plant organic molecules. Recent advances in genomics have opened exciting new perspectives in this field by providing the complete gene inventory of two rhizobial microsymbionts. The complete genome sequences of Mesorhizobium loti, the symbiont of several Lotus species, and Sinorhizobium meliloti, the symbiont of alfalfa, were determined and annotated in detail. For legume macrosymbionts, expressed sequence tag projects and expression analyses using DNA arrays in conjunction with proteomics approaches have identified numerous genes involved in root nodule formation and nitrogen fixation. The isolation of legume genes by tagging or positional cloning recently allowed the identification of genes that control the very early steps of root nodule organogenesis.     

Febrile and acute hyperthermia enhance TNF-induced necrosis of murine L929 fibrosarcoma cells via caspase-regulated production of reactive oxygen intermediates

Previous studies have demonstrated the essential role of TNF-induced reactive oxygen intermediates (ROI) in the necrosis of L929 cells. We investigated the molecular basis for the interaction of hyperthermia and TNF in these cells. Hyperthermia, both febrile (40.0-40.5 degrees C) and acute (41.5-41.8 degrees C), strongly potentiated TNF killing, and sensistization was significantly quenched by the antioxidant, BHA. The broad-spectrum caspase inhibitor, Z-VAD, has been shown to markedly increase the TNF sensitivity of L929 cells at 37 degrees C; we observed that hyperthermia would also enhance the sensitivity of L929 cells to TNF + Z- VAD and that BHA could significantly quench the response, as well. The basis for hyperthermic potentiation was unlikely thermally-increased sensitivity to ROI, as treatment with hydrogen peroxide for 24 h killed L929 cells essentially equivalently, whether incubated continuously at 37 degrees C or at 40.0-40.5 degrees C, or for 2 h at 41.5-41.8 degrees C. However, febrile and acute hyperthermia markedly increased TNF-induced production of ROI, with or without Z-VAD. Hyperthermia dramatically accelerated the onset of this production, as well as the onset of necrotic death, as determined by oxidation of dihydro-rhodamine and propidium iodide staining, respectively, both of which were significantly quenchable with BHA. We conclude that hyperthermia potentiates TNF-mediated killing in this cell model primarily by increasing the afferent, and not the efferent, phase of TNF-induced necrosis.     

Endothelial-like cells derived from human CD14 positive monocytes

In the present study, we show that endothelial-like cells (ELCs) can develop from human CD14-positive mononuclear cells (CD14 cells) in the presence of angiogenic growth factors. The CD14 cells became loosely adherent within 24 h of culture and subsequently underwent a distinct process of morphological transformation to caudated or oval cells with eccentric nuclei. After 1 week in culture the cells showed a clear expression of endothelial cell markers, including von Willebrand factor (vWF), CD144 (VE-cadherin), CD105 (endoglin), acetylated low-density lipoprotein (AC-LDL)-receptor, CD36 (thrombospondin receptor), FLT-1, which is vascular endothelial cell growth factor (VEGF) receptor-1, and, to a weaker extent, KDR (VEGF receptor-2). Furthermore, in these cells structures resembling Weibel-Palade bodies at different storage stages were identified by electron microscopy, and upon culturing on three-dimensional fibrin gels the cells build network-like structures. In addition, cell proliferation and vWF expression was stimulated by VEGF, and the endothelial cell adhesion molecules CD54 (ICAM-1), and CD106 (VCAM-1) became transiently inducible by tumor necrosis factor-alpha (TNF-alpha). In contrast, the dendritic markers CD1a, and CD83 were not expressed to any significant extent. The expression of CD68, CD80 (B7-1), CD86 (B7-2), HLA-DR and CD36 may also suggest that ELCs might be related to macrophages, sinus lining or microvascular endothelial cells. Taken together, our observations indicate that ELCs can differentiate from cells of the monocytic lineage, suggesting a closer relationship between the monocyte/macrophage- and the endothelial cell systems than previously supposed.     

Occurrence of microfilariae in various hollow organs and in the peritoneal cavity after treatment of Litomosoides carinii infected Mastomys natalensis with diethylcarbamazine and haloxon

Treatment of Litomosoides carinii infected Mastomys natalensis with diethylcarbamazine (DEC: 500 mg/kg p.o.) was followed by increased occurrence of microfilariae in the bronchi of the host after 40 min and lasting at least until 6 h after treatment. After 4 h, increased levels of larvae were observed in the gut. Only a few microfilariae occurred in the bladder and sputum. Accumulations of microfilariae were found furthermore in the Lnn. hepaticae whereas no changes were observed in the inguinal or jejunal and lung and pleura associated lymph nodes. Increased numbers of microfilariae were found in the peritoneal cavity only after 8 and continuing until at least 48 h after treatment. In contrast, after haloxon treatment (100 mg/kg p.o.) an accumulation of microfilariae was found in the peritoneal cavity only, following a time course similar to that after DEC.