How does the G protein,Gi2, transduce mitogenic signals?

Serpentine receptors coupled to the heterotrimeric G protein, G_i2, are capable of stimulating DNA synthesis in a variety of cell types. A common feature of the G_i2-coupled stimulation of DNA synthesis is the activation of the mitogen-activated protein kinases (MAPKs). The regulation of MAPK activation by the G_i2-coupled thrombin and acetylcholine muscarinic M₂ receptors occurs by a sequential activation of a network of protein kinases. The MAPK kinase (MEK) which phosphorylates and activates MAPK is also activated by phosphorylation. MEK is phosphorylated and activated by either Raf or MEK kinase (MEKK). Thus, Raf and MEKK converge at MEK to regulate MAPK. G_i2-coupled receptors are capable of activating MEK and MAPK by Raf-dependent and Raf-independent mechanisms. Pertussis toxin catalyzed ADP-ribosylation of α_i2 inhibits both the Raf-dependent and-independent pathways activated by G_i2-coupled receptors. The Raf-dependent pathway involves Ras activation, while the Raf-independent activation of MEK and MAPK does not involve Ras. The Raf-independent activation of MEK and MAPK most likely involves the activation of MEKK. The vertebrate MEKK is homologous to the Ste11 and Byr2 protein kinases in the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The yeast Ste11 and Byr2 protein kinases are involved in signal transduction cascades initiated by pheromone receptors having a 7 membrane spanning serpentine structure coupled to G proteins. MEKK appears to be conserved in the regulation of G protein-coupled signal pathways in yeast and vertebrates. Raf represents a divergence in vertebrates from the yeast pheromone-responsive protein kinase system. Defining MEKK and Raf as a divergence in the MAPK regulatory network provides a mechanism for differential regulation of this system by G_i2-coupled receptors as well as other receptor systems, including the tyrosine kinases.     

Optimization and maintenance of soluble methane monooxygenase activity inMethylosinus trichosporium OB3b

Soluble methane monooxygenase (sMMO) maximization studies were carried out as part of a larger effort directed towards the development and optimization of an aqueous phase, multistage, membrane bioreactor system for treatment of polluted groundwater. A modified version of the naphthalene oxidation assay was utilized to determine the effects of methane:oxygen ratio, nutrient supply, and supplementary carbon sources on maximizing and maintaining sMMO activity inMethylosinus trichosporium OB3b.Methylosinus trichosporium OB3b attained peak sMMO activity (275–300 nmol of naphthol formed h^–1 mg of protein^–1 at 25°C) in early stationary growth phase when grown in nitrate mineral salts (NMS) medium. With the onset of methane limitation however, sMMO activity rapidly declined. It was possible to define a simplified nitrate mineral salts (NMS) medium, containing nitrate, phosphate and a source of iron and magnesium, which allowed reasonably high growth rates (_max 0.08 h^–1) and growth yields (0.4–0.5 g cells/g CH₄) and near maximal activities of sMMO. In long term batch culture incubations sMMO activity reached a stable plateau at approximately 45–50% of the initial peak level and this was maintained over several weeks. The addition of d-biotin, pyridoxine, and vitamin B₁₂ (cyanocobalamin) increased the activity level of sMMO in actively growing methanotrophs by 25–75%. The addition of these growth factors to the simplified NMS medium was found to increase the plateau sMMO level in long term batch cultures up to 70% of the original peak activity.Abbreviations sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - NMS nitrate mineral salts - TCE trichloroethene - NADH reduced nicotinamide adenine dinucleotide     

A common precursor to neurotensin and LANT6 and its differential processing in chicken tissues

Antisera towards neurotensin (NT) and the structurally related peptide, LANT6, were used to characterize immunoreactive peptides and proteins in extracts of chicken tissues. A 17 kDa protein was identified by Western blotting as a potential precursor to NT and LANT6. However, the posttranslational processing of this common precursor appeared to be tissue specific, giving rise to disproportionate amounts of NT and LANT6, along with varying expression of a large molecular LANT6 (M_r, 15 kDa). The intestinal cells containing immunoreactive NT, LANT6, and large molecular LANT6 behaved similarly during fractionation by size and density. These activities also banded together in particles resembling vesicles during centrifugation of isotonic homogenates of tissue. These results suggest that chicken NT and LANT6 are biosynthesized as parts of the same precursor, the processing of which can give rise to a variety of products stored within secretory vesicles.     

Soil factors affecting patchiness in community composition of heavy metal-contaminated areas of Western Europe

The vegetation at various sites within two separate areas (Mechernich and Aachen) of the Eifel Mountains, Germany/Belgium, both characterized by elevated concentrations of heavy metals in their soils, was surveyed in order to investigate the relationships between soil chemical attributes and floristic composition. In both areas, the typical heavy metal communities can form distinct zones, clearly separated from the surrounding heavy metal-sensitive vegetation. However, an intergrading of heavy metal-tolerant and-sensitive vegetation types is not uncommon and such overlaps can occupy large areas. In Mechernich, soil toxicity is primarily determined by the effects of lead, which is best expressed in terms of the Pb/Ca ratio rather than the absolute levels of this metal in the soil. Soils of heavy metal-sensitive vegetation types have a low Pb/Ca ratio, whereas it is considerably higher in areas supporting heavy metal vegetation. Zinc appears to exert little influence on the floristic composition of the investigated vegetation types. In Aachen, zinc is the predominant heavy metal determining vegetation development. Absolute zinc levels of soils do not accurately reflect zinc toxicity. Analogous to the role of the Pb/Ca ratio in the Mechernich area, the Zn/Ca ratio not only separates heavy metal-sensitive and highly tolerant vegetation units, but also gives a good indication of the gradient operating between the two vegetation types. Lead is probably only of local importance in influencing species composition.     

Detection of proteins and sialoglycoproteins in polyacrylamide gels using eosin Y stain

An eosin Y staining technique that permits detection of various proteins, including membrane sialoglycoproteins, in polyacrylamide gels is described. The sensitivity of the eosin Y staining method is comparable to silver staining. In addition, there is an added advantage of the antigen icily of the stained proteins being retained in a Western blot. Details of the procedure to obtain optimal staining results are described.     

Temporal Association Between Pulmonary Inflammation and Antioxidant Induction Following Hyperoxic Exposure of the Preterm Guinea Pig

The time course and nature of the pulmonary inflammatory and antioxidant responses, both during and after hyperoxic-induced acute lung injury were studied in the preterm guinea pig. Three-day preterm (65 days gestation) guinea pigs were randomly exposed to either 21% O₂ (control) or 95% O₂ (hyperoxia) for 72 hours. All pups were then maintained in ambient conditions for up to a further 11 days, during which time lung damage was monitored. In animals exposed to hyperoxia, evidence of acute lung injury and inflammation was characterized by a marked increase in microvascular permeability and elevated numbers of neutrophils in bronchoalveolar lavage fluid. Protein concentration, elastase-like activity and elastase-inhibitory capacity in lavage fluid were at a maximum at the end of the 72 hours hyperoxic exposure. Four days later, all values had returned to control levels. In contrast, increased numbers of neutrophils, macrophages and lymphocytes were recovered in the lavage fluid during this early recovery period. Coinciding with the influx of inflammatory cells, there was a significant increase in glutathione peroxidase, manganese superoxide dismutase and catalase activities in immature lung. Lung copper/zinc superoxide dismutase activity remained unchanged during both experimental periods. The strong temporal relationship between the influx of inflammatory cells to the lung and the induction of pulmonary antioxidant enzyme defences suggests that a common mechanism underlies both responses. These findings have led us to regard inflammation in the hyperoxic-injured immature lung as a beneficial event and not, as previously suggested, as part of the injurious process.     

Individual differences in sensitivity to bitter-tasting substances

Perception of several bitter-tasting compounds was tested in52 subjects. Stable individual differences in the perceivedintensity of the bitterness of suprathreshold concentrationsof quinine sulfate (QSO₄) and urea were found. Whereas 18 subjectsjudged selected concentrations of these compounds to be equallybitter, 17 found QSO₄ to be more bitter than urea, and 17 foundurea to be more bitter than QSO₄. These reliable individualdifferences were significantly related to threshold sensitivityto QSO₄; that is, individuals who perceived QSO₄ to be moreintense than urea at suprathreshold concentrations also hadlower QSO₄ thresholds than did those who perceived urea to bemore intense than QSO₄. There appeared to be no relationshipbetween the relative perceived intensities of these compoundsand rating of the bitterness of PROP (6-n-propylthiouracil).However, QSO₄-sensitive individuals tended to find the bitternessof suprathreshold caffeine and sucrose octaacetate to be greaterthan that of suprathreshold magnesium sulfate, whereas the reversewas true for urea-sensitive individuals. This pattern parallelsthe pattern of cross-adaptation among these compounds reportedby other investigators. These results are consistent with theexistence of multiple bitter transduction sequences and suggestthat individual differences in response to various bitter compoundsmay reflect differences in teh relative availability of specifictransduction sequences.