Continuous culture with complete cell recycle to obtain high cell densities in product inhibited cultures; cultivation ofStreptococcus lactis for production of superoxide dismutase

Summary A cultivation system is described for cultivatingStreptococcus lactis in continuous culture with complete cell recycle. The aim was to obtain high cell densities for the production of su-peroxide dismutase (SOD) while avoiding the growth inhibiting effects of the lactic acid produced. This type of cultivation was performed both at constant and at increasing dilution rates. Comparisons made include those between cell mass productivity and SOD productivity in recycling cultivations and batch cultivations. In the recycling cultivation at increasing dilution rates a cell mass of 19 g/1 was obtained after 22 h of cultivation and the SOD productivity was 43.10³ U/1.h which is four times higher than for batch cultivations. The effect of recyclingS. lactis was also considered and no damage of the microorganisms was observed.     

Characterization of Revertants of Unc-93(e1500) in Caenorhabditis Elegans Induced by N-Ethyl-N-Nitrosourea

Phenotypic reversion of the rubber-band, muscle-defective phenotype conferred by unc-93(e1500) was used to determine the utility of N-ethyl-N-nitrosourea (ENU) as a mutagen for genetic research in Caenorhabditis elegans. In this system, ENU produces revertants at a frequency of 3 X 10(-4), equivalent to that of the commonly used mutagen, EMS. The gene identity of 154 ENU-induced revertants shows that the distribution of alleles between three possible suppressor genes differs from that induced by EMS. A higher percentage of revertants are alleles of unc-93 and many fewer are alleles of sup-9 and sup-10. Three revertants complement the three known suppressor genes; they may therefore identify a new gene product(s) involved in this system of excitation-contraction coupling in C. elegans. Molecular characterization of putative unc-93 null alleles reveals that the base changes induced by ENU are quite different from those induced by EMS; specifically we see an increased frequency of A/T -> G/C transitions. The frequency of ENU-induced intragenic deletions is found to be 13%. We suggest that ENU, at concentrations below 5 mM, will be a superior mutagen for studies of protein function in C. elegans.     

Production of amyloglucosidase by Aspergillus niger under different cultivation regimens

Amyloglucosidase (AMG) was produced by Aspergillus niger in solid-state fermentation (SSF), submerged fermentation (SmF) and an aqueous, two-phase system of polyethyleneglycol (PEG) and salt. In SSF, a fed-batch mode of operation gave a yield of 64 U/ml compared with 44 U/ml in batch mode. Similar trends were observed for SmF, where fed-batch cultivation gave a yield of 102 U/ml compared with 66 U/ml in batch. Shorter cultivation times (66 h) were required for SmF than for SSF (96 h). In the aqueous, two-phase cultivation, the productivity and yield of AMG were both twice those in the control fermentation.M. Ramadas is with the Department of Biochemistry, Faculty of Medicine, University of Jaffna, Kokuvil, Sri Lanka. O. Holst and B. Mattiasson are with the Department of Biotechnology, Chemical Center, Lund University, Box 124, S-221 00 Lund, Sweden     

Stomatal conductance and intrinsic water use efficiency in the drought year 2003: a case study of European beech

Key message

Beech trees were able to cope with the drought of 2003. Harmful water shortage has been avoided by an effective stomatal closure while use of carbon storage pools may have prevented carbon starvation and growth reduction.

Abstract

We applied hydrodynamic modeling together with a tree ring stable isotope approach to identify the physiological responses of beech trees to changing environmental conditions. The drought conditions of the extreme hot and dry summer in 2003 were hypothesized to significantly influence the radial growth of European beech mainly triggered by the stomatal response towards water scarcity leading, in turn, to a decline in carbon assimilation. The functional–structural single tree modeling approach applied, revealed in fact a strong limitation of water use and carbon gain during drought. However, tree ring width data did not show a clear drought response and no differentiation in radial growth during six subsequent years examined (2002–2007) has been observed. Using integrated results from mechanistic carbon–water balance simulations, tree ring carbon and oxygen isotope analysis and tree ring width measurements we postulate that the suggested drought-induced growth decline has been prevented by the remobilization of stored carbohydrates, an early onset in growth and the relatively late occurrence of the severe drought in 2003. Furthermore, we demonstrate that the stomatal response played a significant role in avoiding harmful water tension that would have caused xylem dysfunction. As a result of the combined investigation with physiological measurements (stable isotope approach) and hydrodynamic modeling of stomatal aperture, we could give insights into the physiological control of mature beech tree functioning under drought. We conclude that beech trees have been operating at their hydraulic limits and that the longer or repeated drought periods would have affected the growth considerably.

     

Arabinogalactan Protein from a Crude Cell Organelle Fraction of Phaseolus vulgaris L

Sonication of a crude cell organelle fraction from hypocotyl tissue of dark-grown bean seedlings, and from suspension-cultured cells released a hydroxyproline-containing protein. The purification of this protein is described. It was found to be an arabinogalactan protein composed of 90% carbohydrate and 10% protein. The major sugars are galactose, arabinose, and uronic acids, and the major amino acids are hydroxyproline, serine, and alanine. Its molecular weight was estimated at 1.4 × 10⁵ daltons and the isoelectric point at pH 2.3. The molecule is soluble in 5% trichloroacetic acid and can be precipitated with β-galactosyl Yariv antigen. Pulse-chase experiments indicated that it was a secretory protein. The biosynthesis of arabinogalactan proteins is discussed.     

The heterogeneity of gastric inhibitory polypeptide in porcine and human gastrointestinal mucosa evaluated with five different antisera

The Sephadex G-50 gel filtration profile of immunoreactive gastric inhibitory polypeptide (GIP) in porcine and human gastrointestinal mucosa was determined in assays with antisera obtained from five different groups working with GIP. Tissue was extracted in acid ethanol or using a boiling method. Three well-defined components were detected with three of the antisera: one component corresponding to natural porcine GIP (5 kDa GIP), one component corresponding to what has been called 8 kDa GIP, and one component somewhat larger than the latter. One antiserum did not measure 8 kDa GIP at all, while the fifth antiserum measured small amounts of 8 kDa GIP in porcine but not in human extracts. While the antisera measured the same amounts of GIP in porcine extracts, two of the antisera measured significantly more GIP than the remaining three in extracts of human mucosa. The elution position of human 5 kDa GIP differed significantly from that of porcine 5 kDa GIP. In addition to the identification of a new molecular form of GIP, and the demonstration of important specificity differences among GIP antisera currently in use, the present results indicate that human and porcine 5 kDa GIP differ in chemical composition.     

Localization in the gastrointestinal tract of immunoreactive prosomatostatin

Antisera against 5 different regions of the entire prosomatostatin molecule were used for immunohistochemical mapping of prosomatostatin-containing structures in the pig gastrointestinal tract, and for radioimmunological and chromatographical analysis of the products of prosomatostatin in extracts of ileal mucosa. The latter showed that the antisera were capable of identifying components containing N-terminal as well as C-terminal parts of prosomatostatin. Endocrine cells were identified with all antisera in most parts of the gastrointestinal tract, and varicose nerve fibres were observed in all parts of the small intestine but not in the stomach and the colon. The colon contained very few immunoreactive structures. Immunoreactive nerve cell bodies were found in the submucous plexus of the small intestine. All immunoreactive endocrine cells in the stomach and the duodenum and all immunoreactive nerves were stained by all 5 antisera whereas the small intestinal endocrine cells did not stain for the most N-terminal region of prosomatostatin. The results suggest that all gastrointestinal somatostatin is derived from the same precursor molecule, which, however, in the small intestinal endocrine cells is processed differently from that of the other tissues.     

Innervation of the pancreas by vasoactive intestinal polypeptide (VIP) immunoreactive nerves

The vasoactive intestinal polypeptide (VIP) has been shown to exert effects on endocrine and exocrine pancreatic secretion. Immunocytochemistry reveals that VIP immunoreactive nerves occur in the porcine, canine, feline and avian pancreas. In the pancreas of pig and cat VIP nerves are abundant around non-immunoreactive nerve cell bodies of the intrapancreatic ganglia but scarce in the islets and in the exocrine parenchyma. In the dog pancreas, however, the intrapancreatic ganglia contain strongly immunoreactive VIP nerve cell bodies which give off axons that seem to heavily innervate vessels as well as endocrine and exocrine cells. We suggest that in the pig and cat the pancreatic VIP nerves mainly affect the activity of a second type of intrapancreatic neuron, whose transmitter is unknown, whereas in the dog pancreas VIP nerves directly contact their putative effector structures.