Environmental reprogramming of the expression of protein kinase CK2β subunit in fish

Interleukin-8 (IL-8) is considered as the major polymorphonuclear neutrophils (PMNs) chemoattractant cytokine in lung diseases such as asthma and adult respiratory distress syndrome (ARDS). However, controversial results were obtained regarding the involvement of IL-8 in the pathogenesis of pneumonia. This study examines the role of IL-8 in the recruitment and activation of PMNs in the lung of pneumonia patients. The interesting aspect of this study is that it is a site- specific analysis of the infected and uninfected lungs of the same patient. The level of IL-8 mRNA, protein and myeloperoxidase present in the cells of the bronchioalveolar lavages (BALs) taken from the areas of known pneumonic consolidations on chest X-ray (infected lung) are compared with the BALs obtained from areas of no obvious infiltrate (non-infected lung). The results obtained from the infected and non-infected lungs of pneumonic patients were further compared with that of a control group of non-smoking patients. The level of IL-8 mRNA and protein were determined by RT-PCR and ELISA respectively. There was a significant increase in the level of IL-8 mRNA in the infected lung as compared to its level in the non-infected lung (p < 0.001). In correlation with the increase in mRNA, IL-8 protein concentrations in BAL fluids from the infected lung were 6 fold higher than those taken from the non-infected lung (p < 0.0001). This pattern was also consistent with MPO activity in the BALs (4.5 fold more MPO activity in the infected lung as compared to that of the non-infected lung), indicating that IL-8 is directly implicated in neutrophil accumulation that follows acute respiratory infection. The results of the present study, therefore, indicate the involvement of IL-8 in the pathogenesis of pneumonia.     

5-Hydroxytryptamine2B receptors stimulate Ca2+ increases in cultured astrocytes from three different brain regions

The expression of 5-hydroxytryptamine-2B (5-HT2B) receptor mRNA has recently been shown in cultured astrocytes. Here the expression of functional 5-HT2B receptors has been studied in cultured astrocytes from rat cerebral cortex, hippocampus, and brain stem. Fluo-3- and fura-2-based microspectrofluorometry was used for measuring changes in intracellular free calcium concentrations ([Ca2+]i). The 5-HT2B agonist alpha-methyl 5-HT (40 nM) produced rapid transient increases in [Ca2+]i in astrocytes from all three brain regions studied, and these responses were blocked by the selective 5-HT2B antagonist rauwolscine (1 microM). The specificity of the responses to alpha-methyl 5-HT was further demonstrated by the failure of 4-(4-fluorobenzoyl)-1-(4-phenylbutyl)-piperidine oxalate (1 microM), a specific 5-HT2A/5-HT2C antagonist, to block these responses. The 5-HT2B-induced increases in [Ca2+]i persisted in Ca2+-free buffer, indicating that the increase in [Ca2+]i results from mobilization of intracellular Ca2+ stores. The expression of 5-HT2B receptors on astroglial cells was further verified immunohistochemically and by Western blot analysis. These results provide evidence of the existence of 5-HT2B receptors on astrocytes in primary culture.     

Nonlinkage of major histocompatibility complex class I and class II loci in bony fishes

 In tetrapods, the functional (classical) class I and class II B loci of the major histocompatibility complex (Mhc) are tightly linked in a single chromosomal region. In an earlier study, we demonstrated that in the zebrafish, Danio rerio, order Cypriniformes, the two classes are present on different chromosomes. Here, we show that the situation is similar in the stickleback, Gasterosteus aculeatus, order Gasterosteiformes, the common guppy, Poecilia reticulata, order Cyprinodontiformes, and the cichlid fish Oreochromis niloticus, order Perciformes. These data, together with unpublished results from other laboratories suggest that in all Euteleostei, the classical class I and class II B loci are in separate linkage groups, and that in at least some of these taxa, the class II loci are in two different groups. Since Euteleostei are at least as numerous as tetrapods, in approximately one-half of jawed vertebrates, the class I and class II regions are not linked. Received: 30 August 1999 / Revised: 20 October 1999     

Negative regulation of the serine/threonine kinase B-Raf by Akt

B-Raf contains multiple Akt consensus sites located within its amino-terminal regulatory domain. One site, Ser(364), is conserved with c-Raf but two additional sites, Ser(428) and Thr(439), are unique to B-Raf. We have investigated the role of both the conserved and unique phosphorylation sites in the regulation of B-Raf activity in vitro and in vivo. We show that phosphorylation of B-Raf by Akt occurs at multiple residues within its amino-terminal regulatory domain, at both the conserved and unique phosphorylation sites. The alteration of the serine residues within the Akt consensus sites to alanines results in a progressive increase in enzymatic activity in vitro and in vivo. Furthermore, expression of Akt inhibits epidermal growth factor-induced B-Raf activity and inhibition of Akt with LY294002 up-regulates B-Raf activity, suggesting that Akt negatively regulates B-Raf in vivo. Our results demonstrate that B-Raf activity can be negatively regulated by Akt through phosphorylation in the amino-terminal regulatory domain of B-Raf. This cross-talk between the B-Raf and Akt serine/threonine kinases is likely to play an important role in modulating the signaling specificity of the Ras/Raf pathway and in promoting biological outcome.     

Perfusion system for studying peptide secretion from endocrine cells with high time resolution

To study the secretion from endocrine cells in culture, we have developed a cell column perfusion system with a time resolution of 4 s. The core of the system is a perfusion chamber with a cell-supporting matrix of monosized polystyrene beads. The particles are solid and can withstand a high pressure gradient without deformation. The minimum amount of cell material required to obtain detectable levels of secretory products is a function of the assay sensitivity, perfusion flow, fraction volume and time resolution. The volume of the perfusion chamber is therefore adjustable to satisfy varying demands of minimum cell number. The general flow characteristics of the system were characterized using radiolabeled substances of various molecular sizes. Using the system in secretory studies of rat pituitary tumor (GH4C1) cells, we have identified differences in secretion profiles that may be related to the kinetics of the different transmembrane and intracellular mechanisms involved.     

Homologous and heterologous desensitization and synergy in pathways leading to the soybean oxidative burst

Because the H₂O₂ and O₂ ⁻ generated during a pathogen-triggered oxidative burst could either protect or destroy a besieged plant cell, their synthesis might be expected to be tightly regulated. We have examined the nature of this regulation as it is communicated between homologous and heterologous oxidative-burst pathways, using both chemical (oligogalacturonic acid, harpin, fensulfothion) and mechanical (osmotic stress) stimuli to induce the burst. We report here that the above three chemical elicitors attenuate a subsequent oxidative burst induced in cultured soybean (Glycine max L.) cells by either the same (homologous desensitization) or a different chemical elicitor (heterologous desensitization). Further, when the magnitude of the initial oxidative burst is maximal, the cells remain refractory to subsequent elicitation for at least 10 min and then revive their sensitivities to re-stimulation with a half-time of >20 min. Mechanical stimulation of the oxidative burst appears to be regulated by a different set of constraints. Although initiation of a mechanically induced burst leads to attenuation of a subsequent mechanically induced burst, the same mechanical stimulus is peculiarly unable to reduce a subsequent chemically induced burst. The converse is also true, suggesting that heterologous desensitization of the oxidative burst does not extend to mixed chemical and mechanical/osmotic stimuli. However, communication between these disparate forms of elicitation is still demonstrated to occur, since low-level chemical stimuli strongly synergize concurrent low-level osmotic stimuli and vice versa. Furthermore, the pattern of synergy changes dramatically if one stimulus is administered immediately prior to the other. Taken together, these data demonstrate that significant cross-talk occurs among the different signaling pathways of the oxidative burst and that the overall process is tightly regulated. Received: 10 January 2000 / Accepted: 22 February 2000     

Effects of increased atmospheric CO2 and N supply on photosynthesis, growth and cell composition of the cyanobacterium Spirulina platensis (Arthrospira)

The consequences of the addition of CO2 (1%) in cultures of S. platensis are examined in terms of biomass yield, cell composition and external medium composition. CO2 enrichment was tested under nitrogen saturating and nitrogen limiting conditions. Increasing CO2 levels did not cause any change in maximum growth rate while it decreased maximum biomass yield. Protein and pigments were decreased and carbohydrate increased by high CO2, but the capability to store carbohydrates was saturated. C:N ratio remained unchanged while organic carbon released to the external medium was enhanced, suggesting that organic carbon release in S. platensis is an efficient mechanism for the maintenance of the metabolic integrity, balancing the cell C:N ratio in response to environmental CO2 changes. CO2 affected the pigment content: Phycocyanin, chlorophyll and carotenoids were reduced in around 50%, but the photosynthetic parameters were slightly changed. We propose that in S. platensis CO2 could act promoting degradation of pigments synthetised in excess in normal CO2 conditions, that are not necessary for light harvesting. Nitrogen assimilation was significantly not affected by CO2, and it is proposed that the inability to stimulate N assimilation by CO2 enrichment determined the lack of response in maximum growth rate. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genomic Structure of the Human Gene for Spinocerebellar Ataxia Type 2 (SCA2) on Chromosome 12q24.1

Spinocerebellar ataxia type 2 (SCA2) is a member of a group of neurodegenerative diseases that are caused by instability of a DNA CAG repeat. We report the genomic structure of theSCA2gene. Its 25 exons, encompassing approximately 130 kb of genomic DNA, were mapped onto the physical map of the region. Exonic sizes varied from 37 to 890 bp, and intronic sizes ranged from 323 bp to more than 15 kb. The CAG repeat was contained in the 5′ coding region of the gene in exon 1. Determination of the splice junction sequences indicated the presence of only one deviation from the GT-AG rule at the donor splice site of intron 9, which contained a GC instead of a GT dinucleotide. Exon 10, immediately downstream from this rare splice donor site, was alternatively spliced. Alternative splicing does not affect the reading frame and is predicted to encode an isoform containing 70 amino acids less.     

Ethanol production from native cassava starch by a mixed culture of Endomycopsis fibuligera and Zymomonas mobilis

The conversion of starch from unhydrolyzed cassava flour to ethanol by a pure culture of Endomycopsis fibuligera and by a co-culture of this amylolytic yeast and the bacterium Zymomonas mobilis was studied. The best overall results were obtained using the mixed culture. After 96 h of fermentation of a medium containing 150 g/l initial cassava starch, an ethanol concentration of 31.4 g/l, a productivity of 0.33 g ethanol/l × h and a yield of 0.21 g ethanol/g initial starch were reached. The highest yield (0.37 g/g) was obtained after 48 h when using a medium containing 50 g/l initial starch.