95-kilodalton B-Raf serine/threonine kinase: identification of the protein and its major autophosphorylation site.

B-Raf, a member of the Raf family of serine/threonine kinases, is expressed primarily in the brain and in the nervous system. In this study, the biochemical properties of the B-Raf protein were investigated in nerve growth factor (NGF)-responsive cell lines and in brain tissues. B-Raf was identified by using phosphopeptide mapping analysis and cDNA analysis as a 95-kDa protein which is primarily localized in the cytosol. NGF rapidly stimulated both serine and threonine phosphorylation in vivo and autophosphorylation activity in vitro of the B-Raf protein. In PC12 cells, B-Raf autokinase activity was induced by both differentiation factors and mitogens, with maximal activity observed after 5 min of factor addition. B-Raf kinase activity was also observed following NGF treatment of SH-SY5Y neuroblastoma cells and in adult mouse brain and hippocampus. Induction of B-Raf kinase activity in NGF-treated PC12 cells required expression of kinase-active trk receptors. Exogenous substrates or a peptide containing the autophosphorylation site became phosphorylated when added to immune complex kinase assays and reduced the in vitro autophosphorylation activity of B-Raf, suggesting that in vitro autophosphorylation sites and exogenous substrates compete for active sites of the B-Raf kinase. Finally, the major in vitro autophosphorylation site of B-Raf was identified as threonine 372 in the conserved region 2 domain. A threonine residue is present at similar positions in all three mammalian Raf family members and may represent a regulatory site for these proteins.     

Monitoring of temperature effects on animal cell metabolism in a packed bed process

Animal cell (Chinese Hamster Ovary) concentration was determined on-line in a packed bed process using dielectric spectroscopy. This enabled the evaluation of the effect of temperature on specific metabolic rates during 3 months of continuous culture. The effect of low cultivation temperature on cell growth and metabolism was monitored, and the data were used for process development. At 37 degrees C cells grew exponentially with a specific growth rate of 0.038 d-1 and specific glucose uptake and lactate production rates increased continually. Reduction of the temperature to 33.5 degrees C resulted in a lowering of these metabolic rates while having no effect on cell proliferation. Subsequent reduction of the temperature to 32 degrees C resulted in stabilization of the cell concentration at a high density (3.6 x 10(7) cell per mL of packed bed). In addition, the specific production rate of the protein of interest increased by a factor of 6 compared to the value at 37 degrees C. During the stationary phase at 32 degrees C, all other specific metabolic rates could be controlled to low and constant levels.     

Transforming growth factor-beta: multifunctional regulator of differentiation and development

Transforming growth factors-beta (TGF-beta) are 25 kilodalton (kDa) homodimeric peptides with multifunctional actions controlling the growth, differentiation and function of a broad range of target cells of both epithelial and mesenchymal derivation. They are expressed early in embryogenesis and their tissue-specific and developmentally dependent expression is strongly suggestive of an essential role in particular morphogenetic and histogenetic events. Five distinct TGF-beta s have been characterized so far, with 65-80% homology to each other. By using both molecular biological and immunohistochemical techniques, we are currently attempting to define specific sites of expression of the different TGF-beta s and to determine whether TGF-beta s 1-5 might have unique functions in development and in the mature organism. Comparative study of the promoter regions for the different TGF-beta s and for any particular TGF-beta in different species is also underway. Mechanistically, TGF-beta s act to control gene expression of their target cells, many of their actions converging on a complex, multifaceted scheme of control of matrix proteins and their interactions with cells; these effects on matrix are thought to mediate many of the effects of TGF-beta on development.     

A novel product of the Duchenne muscular dystrophy gene which greatly differs from the known isoforms in its structure and tissue distribution.

In Swiss 3T3 cells, depletion of protein kinase C (PKC) by prolonged incubation with phorbol esters potentiates the formation of total inositol phosphates in response to bombesin or vasopressin [Blakeley, Corps & Brown (1989) Biochem. J. 258, 177-185]. The characteristics of the accumulation of inositol phosphates in control and PKC-depleted cells stimulated by bombesin, vasopressin or prostaglandin F2 alpha (PGF2 alpha) have now been compared. The potentiation of the PGF2 alpha response was greater than that of the vasopressin response which was, in turn, greater than that of the bombesin response. The time courses of the responses to all three agonists were biphasic, and both phases of the response were amplified in the PKC-depleted cells. These results provide further evidence for the involvement of a PKC-mediated negative-feedback loop regulating phosphoinositide hydrolysis in response to several 3T3 cell mitogens. The differential potentiation of the response to these agonists suggests that PKC might act at multiple sites within the signal transduction pathway.     

Association of kidney and parotid Na+, K(+)-ATPase microsomes with actin and analogs of spectrin and ankyrin

Kidney Na+,K(+)-ATPase has been recently shown to bind erythroid ankyrin and to colocalize with ankyrin at the basolateral cell surface of kidney epithelial cells. These observations suggest that Na+,K(+)-ATPase is linked via ankyrin to the spectrin/actin-based membrane cytoskeleton. In the present study we show that Na+,K(+)-ATPase and analogs of spectrin, ankyrin and actin copurify from detergent extracts of pig kidney and parotid gland membranes. Actin, spectrin and ankyrin were extracted from purified Na+,K(+)-ATPase microsomes at virtually identical conditions as their counterparts from the erythrocyte membrane, i.e., 1 mM EDTA (spectrin, actin) and 1 M KCl (ankyrin). Visualization of the stripped proteins by rotary shadowing revealed numerous elongated spectrin-like dimers (100 nm) and tetramers (215 nm), a fraction of which (17%) was associated with globular (10 nm) ankyrin-like particles. Like erythrocyte ankyrin, kidney ankyrin was cleaved into a soluble 72 kDa fragment and a membrane-bound 90 kDa fragment. Consistent with our previous immunocytochemical findings on the pig kidney, Na+,K(+)-ATPase and ankyrin were found to be colocalized at the basolateral plasma membrane of striated ducts and acini of the pig parotid gland. The present findings confirm and extend the recently proposed concept that in polarized epithelial cells Na+,K(+)-ATPase may serve as major attachment site for the spectrin-based membrane cytoskeleton to the basolateral cell domain. Connections of integral membrane proteins to the cytoskeleton may help to place these proteins at specialized domains of the cell surface and to prevent them from endocytosis.     

The predator guild associated withAceria litchii (Acari: Eriophyidae) in Australia and China

Lychees were surveyed in Queensland, Australia and in Guangdong Province and Hainan Island China, for natural enemies of the lychee erinose mite,Aceria litchii (Keifer), one of the most serious pests of lychee in Australia. A guild of seventeen predators, including ten species of phytoseiid mites, was associated with lychee erinose in Queensland. Six other predaceous mite species and a cecidomyiid larva,Arthrocnodax sp. were also part of the complex. Despite the apparent predation of most of the seventeen species recorded in Queensland onA. litchii, the pest continues to cause major problems. In China, whereA. litchii is a relatively minor pest, nine phytoseiid species were collected in lychee orchards. The value of introducing additional predators to Australia, especially from China, is discussed.     

A novel action of glucocorticosteroid in inhibition of leukotriene C4 production by rat basophilic leukemia cells: suppression of the elevation of cytosolic free Ca2+ induced by antigen

Rat basophilic leukemia (RBL-2H3) cells serve as a model to examine the role of elevated internal Ca2+ concentration ([Ca2+]i), following antigen (DNP10BSA)-induced stimulation of leukotriene C4 (LTC4) formation. A novel action of hydrocortisone (HC), to reduce increased [Ca2+]i and consequently inhibit LTC4 formation is assessed. Half-maximal time for elevation of [Ca2+]i induced by antigen was less than 1 min, and maximal elevation of [Ca2+]i (3-fold increase) was reached within 2-3 min. This high [Ca2+]i level waned gradually by 27% during 20 min of incubation. For induction of LTC4 formation, however, there was a refractory period of about 2 min, and half-maximal elevation was at 11 min. Following pretreatment with HC, the antigen-stimulated increase in [Ca2+]i was stunted by 41% at 2-3 min and by 73% at 20 min. LTC4 formation was almost abolished. There was a lag period of at least 2 h to observe any inhibition in both parameters, and the maximal inhibition was about 4 h. Cycloheximide, and receptor antagonist to glucocorticosteroid (RU486) completely prevented the inhibitory effects of HC on elevated [Ca2+]i and LTC4 formation. Estradiol and aldosterone (each at 2.10(-6) M) were virtually inactive, while another glucocorticosteroid, dexamethasone (2.10(-7) M) markedly suppressed antigen induction in both parameters. It is proposed that the inhibitory effect of HC on the formation of LTC4 could be attributed mainly to its ability to reduce elevated [Ca2+]i.