Epidermal Growth Factor Inhibits Gap Junctional Communication and Stimulates Serine-Phosphorylation of Connexin43 in WB Cells by a Protein Kinase C-Independent Mechanism

Epidermal growth factor (EGF) stimulated the phosphorylation of connexin43 (Cx43) in WB cells as evidenced by the formation of multiple irnmunoreactive Cx43 proteins of higher molecular mass which were abolished by treatment with alkaline phosphatase. Phosphorylation of Cx43 occurred within 10 min of EGF stimulation, was sustained for 1 h, and was associated with almost complete inhibition of gap junctional communication in these cells. EGF-induced phosphorylation and communication inhibition were retained in cells pretreated with phorbol 12-myristate 13-acetate (PMA) to deplete protein kinase C. These results show that the EGF inhibition of communication is tightly linked to protein kinase C-independent phosphorylation of Cx43. Further, Cx43 phosphorylated in the presence of EGF did not react with phosphotyrosine antibodies and in ³²P_i incorporation experiments was shown to contain only phosphoserine indicating that the tyrosine kinase activity of the EGF receptor was not directly involved.     

New branched Porolithon species (Corallinales,Rhodophyta) from the Great Barrier Reef,Coral Sea,and Lord Howe Island

Porolithon is one of the most ecologically important genera of tropical and subtropical crustose (non-geniculate) coralline algae growing abundantly along the shallow margins of coral reefs and functioning to cement reef frameworks. Thalli of branched, fruticose Porolithon specimens from the Indo-Pacific Ocean traditionally have been called P. gardineri, while massive, columnar forms have been called P. craspedium. Sequence comparisons of the rbcL gene both from type specimens of P. gardineri and P. craspedium and from field-collected specimens demonstrate that neither species is present in east Australia and instead resolve into four unique genetic lineages. Porolithon howensis sp. nov. forms columnar protuberances and loosely attached margins and occurs predominantly at Lord Howe Island; P. lobulatum sp. nov. has fruticose to clavate forms and free margins that are lobed and occurs in the Coral Sea and on the Great Barrier Reef (GBR); P. parvulum sp. nov. has short (<2 cm), unbranched protuberances and attached margins and is restricted to the central and southern GBR; and P. pinnaculum sp. nov. has a mountain-like, columnar morphology and occurs on oceanic Coral Sea reefs. A rbcL gene sequence of the isotype of P. castellum demonstrates it is a different species from other columnar species. In addition to the diagnostic rbcL and psbA marker sequences, the four new species may be distinguished by a combination of features including thallus growth form, margin shape (attached or unattached), and medullary system (coaxial or plumose). Porolithon species, because of their ecological importance and sensitivity to ocean acidification, need urgent documentation of their taxonomic diversity.     

Importance of spore mutants for fed-batch and continuous fermentation of Bacillus subtilis

To alleviate plasmid instability and to prolong the production phase of subtilisin, integrable plasmid and spore mutants are used. Compared with batch-type shake flask cultures, spore mutants' ability to produce subtilisin can be well pronounced in fed-batch and continuous cultures. Hence, the two culture methods make it possible to identify the peculiar characteristics of the spore mutants unobtainable in batch culture. Spore mutants can enhance subtilisin productivity and prolong subtilisin production time in fed-batch culture as well as enable us to use very low dilution rates (<0.1 h(-1)) without losing productivity in continuous culture, thereby improving the conversion yield of the nitrogen source. At 0.05 h(-1) the spollG mutant of Bacillus subtilis DB104 (Deltanpr Deltaapr) (Em(r)) spollG (Bim(r)):: pMK101 (Cm(r)) showed a subtilisin yield about ten times higher than that from wild-type DB104 (Deltanpr Deltaapr)::pMK101 (Cm(r)). (c) 1995 John Wiley & Sons, Inc.     

Mutational analysis of the three cysteines and active-site aspartic acid 103 of ketosteroid isomerase from Pseudomonas putida biotype B.

In order to clarify the roles of three cysteines in ketosteroid isomerase (KSI) from Pseudomonas putida biotype B, each of the cysteine residues has been changed to a serine residue (C69S, C81S, and C97S) by site-directed mutagenesis. All cysteine mutations caused only a slight decrease in the k(cat) value, with no significant change of Km for the substrate. Even modification of the sulfhydryl group with 5,5'-dithiobis(2-nitrobenzoic acid) has almost no effect on enzyme activity. These results demonstrate that none of the cysteines in the KSI from P. putida is critical for catalytic activity, contrary to the previous identification of a cysteine in an active-site-directed photoinactivation study of KSI. Based on the three-dimensional structures of KSIs with and without dienolate intermediate analog equilenin, as determined by X-ray crystallography at high resolution, Asp-103 was found to be located within the range of the hydrogen bond to the equilenin. To assess the role of Asp-103 in catalysis, Asp-103 has been replaced with either asparagine (D103N) or alanine (D103A) by site-directed mutagenesis. For D103A mutant KSI there was a significant decrease in the k(cat) value: the k(cat) of the mutant was 85-fold lower than that of the wild-type enzyme; however, for the D103N mutant, which retained some hydrogen bonding capability, there was a minor decrease in the k(cat) value. These findings support the idea that aspartic acid 103 in the active site is an essential catalytic residue involved in catalysis by hydrogen bonding to the dienolate intermediate.     

Replating of bioreactor expanded human bone marrow results in extended growth of primitive and mature cells

The capability to expand human bone marrow mononuclear cells (BM MNC) in high density perfusion culture chambers (bioreactors) has recently been developed. In these bioreactors, total cell colony-forming unit-granulocyte/macrophage (CFU-GM), and long-term culture-initiating cell (LTC-IC) numbers increase significantly over a 14-day period. However, cell growth ceases after the 14-day period, possibly due to cell density limitations. Because of the remaining presence of early cells, it should be feasible to replate the cells and obtain continued expansion. In this study, we demonstrate that bioreactors generate cells, which upon replating into secondary bioreactors, lead to continued cell, CFU-GM, and LTC-IC₈ (measured after 8 weeks of secondary culture) expansion. A two-stage protocol, involving the replating of cells on days 9 to 12 of culture into new bioreators at the original seeding density, yielded greater than 50-fold cell expansion from BM MNC in 25 days. CFU-GM were expanded inhibitory factor (LIF) had no significant effect on total cells, CFU-GM, or LTC-IC₅ in this system. We conclude that two-stage bioreactor cultures are capable of supporting extended growth of human BM MNC, CFU-GM, and LTC-IC₈. The continued expansion of these primitive cells in the second stage of culture suggests that primitive cells with significant proliferative potential were generated in this system, and previous data on LTC-IC₅ expansion has now been extended to LTC-IC₈ expansion. Further optimization of culture conditions is likely to improve on the results obtained here, thus making perfusion bioreactor culture correspondingly more attractive for expanding BM MNC for BM transplantation.     

Comparative structural study ofQuercus serrata andQ. acutissima formed byPisolithus tinctorius andHebeloma cylindrosporum

Ectomycorrhizas were synthesized in pots and growth pouches betweenQuercus serrata, Q. acutissima, and two ectomycorrhizal fungi,Pisolithus tinctorius andHebeloma cylindrosporum. Root morphology and the structure of the mantle and Hartig net were compared using light, fluorescence, scanning and transmission electron microscopy.P. tinctorius initially colonized root cap cells, and eventually produced a highly branched lateral root system with a complete mantle, whereasH. cylindrosporum promoted root elongation with few hyphae on the root apex surface indicating that interaction between roots differs with fungal species. Hartig net structure and hyphal inclusions varied between all the combinations tested. There were structural differences between mycorrhizas ofH. cylindrosporum/Q. acutissima grown in soil and growth pouches, which indicate that the growth pouch environment can induce artefacts in roots. Fruit bodies ofH. cylindrosporum developed in pots withQ. acutissima. AlthoughP. tinctorius has been used to inoculate oak seedlings in the nursery, results of this study indicate thatH. cylindrosporum may also be an effective ectomycorrhizal fungus forQ. serrata andQ. acutissima.     

Neurotrophic control of cyclic nucleotide levels during muscle differentiation in cell culture

The effects of chick brain–spinal cord extract on morphological development and cyclic nucleotide levels of cultured chick embryo skeletal muscle cells were determined. It had previously been shown that the extract stimulated morphological differentation, protein synthesis, and cholinesterase activity of muscle cells. Myoblasts fused earlier and an increase in number as well as diameter of myotubes were seen in the extract treated cultures. Cyclic nucleotides levels were higher (almost twice the controls for both adenosine 3′, 5′ -cyclic monophosphate and guanosine 3′, 5′ -cyclic monophosphate) and preceded their occurence in the control cultures. It was suggested that factor(s) in the extract interact with membrane receptor(s) to alter nucleotide levels which, in turn, allow the effects to be expressed.