Increased podophyllotoxin production in Podophyllum hexandrum cell suspension cultures after feeding coniferyl alcohol as a β-cyclodextrin complex

Summary Cell suspension cultures, derived from roots of Podophyllum hexandrum Royle (Berberidaceae), accumulate podophyllotoxin. In this study the use of -cyclodextrin in feeding the poorly water-soluble precursor coniferyl alcohol to these cultures is described. By complexation with -cyclodextrin, a solution of 3 mM coniferyl alcohol could be fed, resulting in enhanced podophyllotoxin accumulation. The same concentration of non-complexed suspended coniferyl alcohol had only little effect on the podophyllotoxin accumulation. -Cyclodextrin itself was proven to be non-toxic for the cells. It did not influence the podophyllotoxin content and it was not metabolized or used as a carbon source by the cells. For comparison, coniferin, the water-soluble -D-glucoside of coniferyl alcohol, was also fed in the same concentration. The effect of coniferin on the podophyllotoxin accumulation was stronger than that of coniferyl alcohol complexed with -cyclodextrin, but coniferin is not commercially available.Abbreviations -CD -cyclodextrin - NAA naphthaleneacetic acid     

Expression of the FGF-related proto-oncogene int-2 during gastrulation and neurulation in the mouse.

The proto-oncogene int-2 has been implicated in the formation of mouse mammary-tumour-virus-induced mammary tumours. Analysis of the predicted coding sequence indicates that int-2 is a member of the fibroblast growth factor family. Previous studies using Northern blot analysis suggested that normal expression of int-2 may be confined to extra-embryonic endoderm lineages of embryonic stages of mouse development. We have used in situ hybridization and Northern blot analysis to examine directly int-2 expression in embryo stem cells and in the developing embryo from early gastrulation to midsomite stages. Complex patterns of accumulation of int-2 RNA were observed in embryonic and extra-embryonic tissues. The data suggest multiple roles for int-2 in development which may include migration of early mesoderm cells and induction of the otocyst.     

Restriction pattern and polypeptide homology among plasmid-borne mercury resistance determinants

The structural and functional properties of mercury resistance determinants cloned from a series of independently isolated conjugative plasmids were compared with those of the prototype HgR determinants from Tn501 and plasmid R100 (containing Tn21). Restriction endonuclease mapping classified the HgR determinants into at least three different but related structural groups which are distantly related to those from Tn501 and R100. These relationships were confirmed by the functional analysis of sub-clones and gamma delta insertion mutations and from the polypeptides specified by the cloned HgR determinants. Each mercury resistance clone synthesized polypeptides equivalent in size to the merA, merT, and merP gene products. However, those for merA and merT showed considerable size variation. No polypeptide equivalent to merD or merC of R100 was detected.     

Inverse relationship between proliferation and differentiation in a human TNP-specific B cell line. Cell cycle dependence of antibody secretion

Studies of an EBV-transformed and TNP-specific human B cell line revealed that, unlike myeloma or hybridoma cell lines that consist mainly of fully differentiated cells, most of the cloned EBV-transformed cells were not fully differentiated, as judged by inability to bind TNP-SRBC and to secrete anti-TNP antibody. The minority of more differentiated cells were selected by TNP-SRBC rosetting. They were found to proliferate to a lesser extent than nonrosetting cells and to contain increased numbers of antibody-secreting cells. This inverse relationship between proliferation and differentiation was also shown to be cell cycle related in that the TNP-SRBC rosetting cells resided, to a greater extent than the nonrosetting cells, in the G1 phase of the cell cycle. The finding that the G1 phase of the cell cycle was associated with differentiation into anti-TNP secreting cells was confirmed by demonstrating that treatment with hydroxyurea, which arrests the cells in G1, resulted in decreased proliferation and an increased proportion of antibody-secreting cells. Similarly, addition of phorbol ester resulted in increased antibody secretion and decreased proliferation, suggesting a role for protein kinase C in this differentiation pathway. The strategy of increasing the number of antibody-producing cells in this human EBV line, by promoting differentiation of the cells in G1, may be relevant to the large scale production of specific human mAb for the treatment and diagnosis of human diseases.     

Biochemical analysis of the activation of adherent neutrophils in vitro

The role played by neutrophil oxidative responses in host defense and injury is an area of active investigation. In order to study neutrophil responses in vitro, methods are required for cell purification, enumeration, and quantification of activation responses, which mimic the in vivo situation as closely as possible. In this communication (and its companion paper, Albertine et al., 1988) improved methods for all of these tasks are described and applied to investigate neutrophil structure-function relationships in vitro and in vivo. Human neutrophils were purified by using a series of platelet-poor plasma-Percoll gradients (51, 62, 76 and 80% in Percoll). This modification of previously published procedures results in consistently successful neutrophil purification and has allowed us to purify neutrophils from bronchoalveolar lavage fluid as well as blood. Activation of human and sheep neutrophils (superoxide anion production) was quantitated by the reduction of ferricytochrome c using a microtiter plate reader to measure the increase in absorbance at 550 nm from adherent neutrophils. Adherence of neutrophils was quantitated by measurement of LDH in cells lysed with Triton X-100 using a new method which uses readily available commercial reagents and can quantitate the LDH content of as few as 5000 neutrophils (or the LDH released from 5% of 100,000 neutrophils). Assay conditions for superoxide anion were optimized, limitations both in assay design and instruments used to measure OD were explored and enumerated, and these methods were used to quantitate sheep and human neutrophil activation responses. Using methods described in Albertine et al. (1988) for fixing neutrophils in microtiter wells after assay of their functional capacity, we have studied the same cells functionally and morphologically. We have used these techniques to study blood and alveolar neutrophils from a patient with acute respiratory failure. His alveolar neutrophils displayed 67% of the activation response as peripheral neutrophils (4.31 +/- 0.12 nmol superoxide released per 250,000 neutrophils at 60 min vs. 6.38 +/- 0.18 in blood, P less than 0.01) and structural changes which suggested previous activation in vivo. These studies demonstrate that similar morphological changes are observed in neutrophils activated with phorbol myristate acetate in vitro, as are observed in cells which have been activated by pathophysiologic processes in vivo.     

A method for online-estimation of cellulose concentration in a fermentation process

Summary A method based on the elemental balance of the process has been developed to calculate the consumption of cellulose in cellulase production byTrichoderma reesei Rut-C30. Using a synthetic medium only the oxygen in effluent gas and consumption of base for pH-correction has to be measured.     

Studies on the System Regulating Proton Movement across the Chloroplast Envelope : Effects of ATPase Inhibitors, Mg, and an Amine Anesthetic on Stromal pH and Photosynthesis

Studies were undertaken to further characterize the spinach (Spinacea oleracea) chloroplast envelope system, which facilitates H⁺ movement into and out of the stroma, and, hence, modulates photosynthetic activity by regulating stromal pH. It was demonstrated that high envelope-bound Mg²⁺ causes stromal acidification and photosynthetic inhibition. High envelope-bound Mg²⁺ was also found to necessitate the activity of a digitoxinand oligomycin-sensitive ATPase for the maintenance of high stromal pH and photosynthesis in the illuminated chloroplast. In chloroplasts that had high envelope Mg²⁺ and inhibited envelope ATPase activity, 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide was found to raise stromal pH and stimulate photosynthesis. 2-(Diethylamino)-N-(2,6-dimethylphenyl)acetamide is an amine anesthetic that is known to act as a monovalent cation channel blocker in mammalian systems. We postulate that the system regulating cation and H⁺ fluxes across the plastid envelope includes a monovalent cation channel in the envelope, some degree of (envelope-bound Mg²⁺ modulated) H⁺ flux linked to monovalent cation antiport, and ATPase-dependent H⁺ efflux.     

Ultrastructure of the approximately 26S complex containing the approximately 20S cylinder particle (multicatalytic proteinase/proteasome).

We have isolated a large protein complex of approximately 26S from Xenopus laevis oocytes and eggs which is composed of the approximately 20S cylinder particle (multicatalytic proteinase/proteasome) and additional proteinaceous components. In its polypeptide composition and sedimentation coefficient this approximately 26S complex closely resembles the 26S ubiquitin-dependent protease, a high molecular weight multienzyme complex recently described in the literature. Specific antibodies directed against a single subunit of the approximately 20S cylinder particle retain, on affinity columns, the large approximately 26S complex, and on sucrose gradients up to approximately 50% of the approximately 20S cylinder particles present in oocyte extracts sedimented with approximately 26S, suggesting that a large proportion of the approximately 20S particles exists in the cell as a component of the approximately 26S complex. Electron microscopy reveals the approximately 26S complex to be a symmetrical elongated macromolecular assembly of at least three protein particles. The central core of the complex is formed by the approximately 20S cylinder particle to which two other large components are attached at the ends, yielding a dumbbell-shaped complex of approximately 40 nm in length. Dissociation of the approximately 26S complexes releases in addition to approximately 20S cylinder particles a novel type of a disc-shaped particle of approximately 15 nm diameter which may represent the attached components or subcomplexes of them. Based on its structural and biochemical properties we postulate that the approximately 26S complex identified here is identical to the ubiquitin-dependent protease.     

Murine erythrocyte ankyrin cDNA: highly conserved regions of the regulatory domain

Ankyrin is an essential link between cytoskeletal proteins, such as spectrin, and membrane bound proteins, such as protein 3, the erythrocyte anion exchanger. Although the amino acid structure of human ankyrin is known, the functional regions have been only partially defined. Sequence comparisons between mouse and human ankyrin offer one mechanism of identifying highly conserved regions that probably have functional significance. We report the isolation and sequencing of a series of overlapping murine erythroid ankyrin (Ank-1) cDNAs from spleen and reticulocyte libraries (total span 6238 bp) and identify potentially important regions of murine-human reticulocyte ankyrin homology. Comparison of the predicted peptide sequences of mouse and human erythroid ankyrins shows that these ankyrins are highly conserved in both the N-terminal, protein 3 binding domain (96% amino acid identity) and in the central spectrin-binding domain (97% identity), but differ in the C-terminal regulatory domain (79% identity). However, the C-terminal regulatory domain contains two regions of peptide sequence that are perfectly conserved. We postulate these regions are important in the regulatory functions of this domain.