Production of alkaline phosphatase by immobilized growing cells of Escherichia coli excretory mutants

Summary In continuous cultures, alkaline phosphatase was synthesised and excreted for more than 250 h by immobilized growing cells in contrast to free cells for which the excretion decreased after 150 h of culture. This observed increase in alkaline phosphatase synthesis and excretion by immobilized cells may have resulted from growing conditions within the gel beads.Offprint requests to: C. Manin     

Continuous production of gibberellic acid in a fixed-bed reactor by immobilized mycelia of Gibberella fujikuroi in calcium alginate beads

Summary The continuous production of gibberellic acid with immobilized mycelia of Gibberella fujikuroi was maintained over a hundred days in a tubular fixed-bed reactor. Free mycelium at the beginning of the storage phase was harvested from G. fujikuroi shake-flask culture and was immobilized by ionotropic gelation in calcium alginate beads.The continuous recycle production system consisted of a fixed-bed reactor, a container in which the culture medium was heated, stirred and aerated, and valves for sample withdrawal or reactant addition during the first 1320 h (55 days). A two-phase continuous extractor was then added for the last 960 hours (40 days). Free and immobilized mycelium shake-flask cultures with the same strain used in the continuous culture system were also realized to compare growth, maintenance and production parameters. The results show about the same gibberellic acid productivity in both free and immobilized mycelium shakeflask cultures: 0.384 and 0.408 mgGA₃·gBiomass^-1 ·day^-1, respectively, whereas in the continuous system the gibberellic acid production is about twice as large for a similar biomass: 0.768 mgGA₃·gBiomass^-1·day^-1. Several factors affecting the overall productivity of the immobilized systems were found to be: the quality and the quantity of mycelia in the biocatalyst beads and the immobilization conditions.     

Insulin binding in the hypothalamus of lean and genetically obese Zucker rats

Recent reports have suggested that the obesity and hyperphagia of the genetically obese Zucker rat may be related to defective insulin action or binding in the hypothalamus. We used quantitative autoradiography to determine if insulin binding is altered in specific hypothalamic nuclei associated with food intake. Insulin binding was measured in the arcuate (ARC), dorsomedial (DMN), and ventromedial (VMN) hypothalamic nuclei of 3–4-month-old lean (Fa/Fa) and genetically obese (fa/fa) Zucker rats. A consistently reproducible 15% increase in the total specific binding of 0.1 nM [¹²⁵I]-insulin was found in the ARC of the obese genotype. A slight increase in insulin binding in the DMN was also found. No difference in specific insulin binding was found between genotypes in the VMN. Nonlinear least squares analysis of competitive binding studies showed that the K_d of the ARC insulin binding site was 33% higher in the lean rats than in the obese rats, indicating an increased affinity for insulin. No difference in site number (B_max) was found in the ARC, DMN or VMN, and no evidence was found for reduced insulin binding in the hypothalamus of the obese (fa/fa) genotype. The results suggest that hyperphagia and obesity of the obese (fa/fa) Zucker rat genotype may be associated with increased insulin binding in the arcuate nucleus.     

Regulation of branched-chain amino acid biosynthesis in Streptomyces fradiae,a producer of tylosin

Synthesis of threonine dehydratase in Streptomyces fradiae was positively influenced by valine and negatively by isoleucine. However, these two amino acids had no effect on the activity of this enzyme. Synthesis of threonine dehydratase in -aminobutyrate resistant mutants of S. fradiae was pronouncedly less sensitive to the positive effect of valine and this change in regulation led to valine overproduction. Synthesis of acetohydroxy acid synthase is regulated in a similar manner to that of threonine dehydratase, however a lower level of expression was detected in -aminobutyrate resistant mutants. And again, no effect of branched-chain amino acids on acetohydroxy acid synthase activity was observed. It follows that in S. fradiae synthesis of threonine dehydratase is the main regulatory mechanism governing production and the mutual ratio of synthesized valine and isoleucine.Abbreviations -AB -aminobutyrate - AHAS acetohydroxy acid synthase - -KB -ketobutyrate - MNNG N-methyl-N-nitro-N-nitrosoguanidine - TD threonine dehydratase - Trans. B. transaminase of branched-chain amino acids - VDH valine dehydrogenase     

Does in vitro activation of postsynaptic alpha 2-adrenoceptor utilize intracellular Ca2+ for contraction in dog saphenous vein?

Dog saphenous vein spiral strips were employed to determine whether an intracellular source of Ca2+ is used for contraction upon activation of the alpha 2-adrenoceptor by B-HT 920 in Ca2+-free Krebs solution containing 50 microM EGTA. The studies were carried out in parallel with the activation of the alpha 1-adrenoceptor by phenylephrine (Phe) under the condition that B-HT 920 (10(-5) M) and Phe (2 x 10(-6) M) gave rise to a similar level of responses in Ca2+-containing Krebs solution. A similar level of responses to these agonists at equieffective concentrations in Ca2+-free medium were also observed. Such responses to Phe and B-HT 920 were inhibited by 10(-7) M rauwolscine and 10(-7) M prazosin, respectively, and were not affected by 10(-7) M nifedipine or 5 mM Mn2+. The responses to B-HT 920 (10(-5) M) and submaximal concentration of Phe (2 x 10(-6) M) in Ca2+-free medium were additive. However, if the vascular strips were first contracted maximally with 10(-4) M Phe in Ca2+-free medium to deplete the intracellular Ca store, subsequent addition of B-HT 920 failed to induce additional response. Our results strongly suggest that activation of alpha 2-adrenoceptor in dog saphenous vein in Ca2+-free medium indeed utilizes intracellular Ca2+ for contraction. We also found that the failure of earlier studies to demonstrate the contractile effects of B-HT 920 in dog saphenous vein was due to experimental artifacts derived from the use of high concentration of EGTA and artificial pH-buffering reagent.     

Extrachromosomal circular DNAs in Drosophila melanogaster: Comparison between embryos and Kc0% cells

We established the size distribution of extrachromosomal covalently closed circular DNA molecules from embryos of various Drosophila melanogaster strains and from Kc0% tissue culture cells. In embryos, more than 80% of the circular DNA molecules are smaller than 2.5 kb and all the distributions show a peak of molecules of between 200 and 400 bp. The Kc0% cell distribution differs mainly from that of embryos in that 48% of the molecules have a size between 4 and 8 kb. Correlating with this, circular molecules homologous to copia, 412 and 297 were detected only in Kc0% cells. The three tandemly repeated families containing the 5S genes, the histone genes and the 240 bp repeat of the ribosomal DNA intergenic spacer, which had previously been identified in circular DNAs from embryos, were also found in cultured cells. A fourth tandemly repeated family corresponding to the 1.688 g/cm³ satellite DNA was detected, both in embryos and Kc0% cells. It consists of circular multimeric molecules containing multiple copies of the 359 bp repeated unit. No circular DNA molecules homologous to the actin genes, the type I ribosomal DNA insertion, or the F and I transposable elements were found in embryos or Kc0% cells. Thus it appears that the extrachromosomal circular DNA molecules from embryos and from tissue culture cells differ mainly in the presence of circular copies of the copia-like transposable elements.     

TGF-beta 1-induced inhibition of mouse mammary ductal growth: developmental specificity and characterization

TGF-beta 1, implanted into growing mouse mammary glands, was previously shown to inhibit ductal growth in an apparently normal and fully reversible manner. In this report we extend these findings to show that TGF-beta 1 inhibition is highly specific. In pregnant or hormone-treated mice, doses of TGF-beta 1 that were capable of fully inhibiting ductal elongation had little effect on the proliferation of lobuloalveolar structures. Additionally, the inhibitory action of TGF-beta 1 on ducts is epithelium-specific, resulting in cessation of DNA synthesis in the rapidly proliferating epithelium of mammary end buds, but does not inhibit DNA synthesis in the stroma surrounding the end buds. At the cellular level, transplant studies showed that TGF-beta 1 inhibited the regeneration of mammary ductal cells when implanted into mammary gland-free fat pads by suppressing the formation of new end buds, without inhibiting maintenance DNA synthesis in ductal lumenal epithelium; this observation indicates the potential of TGF-beta 1 to maintain patterning by suppressing adventitious lateral branching. The time-course of TGF-beta 1 inhibition of end buds was rapid, with cessation of DNA synthesis by 12 hr, followed by loss of the stem cell (cap cell) layer. The question of glandular exposure to TGF-beta 1 administered in EVAc implants was also investigated. Incorporation of TGF-beta 1 into EVAc was found not to degrade the hormone, while the release kinetics of the ligand from implants, its retention in the gland, and the demonstrable zone of exposure were consistent with observed inhibitory effects. These results support the hypothesis that TGF-beta 1 is a natural regulator of mammary ductal growth.     

Cytochalasin D inhibits basal body migration and ciliary elongation in quail oviduct epithelium

Summary The effects of cytochalasin D (CD) were studied by scanning (SEM) and transmission (TEM) electron-microscopic examination at different stages of ciliary differentiation in epithelial cells of quail oviduct. Immature quails were prestimulated by estradiol benzoate injections to induce ciliogenesis in the undifferentiated oviduct. After 24 h of CD culture, SEM study revealed inhibition of ciliogenesis and dilation of the apex of non-ciliated cells. TEM study showed that 2 h of CD treatment produced dilation of lateral intercellular spaces, after 6 h of treatment, this resulted in intracellular macrovacuolation. Vacuoles were surrounded by aggregates of dense felt-like material. CD also induced the disappearance of microvilli, and rounding of the apical surface of undifferentiated cells and those blocked in ciliogenesis. Centriologenesis was not inhibited by CD; basal bodies assembled in generative complexes in the supranuclear region after 24 h of treatment. However, the migration of mature basal bodies towards the apical surface was impaired. Instead, they anchored onto the membrane of intracellular vacuoles; growth of cilia was induced in the vacuole lumen. Cilium elongation was disturbed, giving abnormally short cilia with a dilated tip; microtubules failed to organize correctly.     

Patterns of [13N]ammonium uptake and assimilation by Frankia HFPArl3

Nitrogen-starved cells of Frankia strain HFPArl3 incorporated [¹³N]-labeled ammonium into glutamine serine (glutamate, alanine, aspartate), after five-minute radioisotope exposures. High initial endogenous pools of glutamate were reduced, while total glutamine increased, during short term NH _inf4 ^sup+ incubation. Preincubation of cells in methionine sulfoximine (MSX) resulted in [¹³N]glutamine reduced by more than 80%, while [¹³N]glutamate and [¹³N]alanine levels increased. The results suggest that glutamine synthetase is the primary enzyme of ammonium assimilation, and that glutamate dehydrogenase and alanine dehydrogenase may also function in ammonium assimilation at low levels. Efflux of [¹³N]serine and lesser amounts of [¹³N]glutamine was detected from the Frankia cells. The identity of both Ser and Gln in the extracellular compartment was confirmed with gas chromatography/mass spectrometry. Serine efflux may be of significance in nitrogen transfer in Frankia.Abbreviations Pthr phosphothreonine - Aad -amino-adipate - MSX methionine sulfoximine     

Circadian locomotor rhythms in the desert iguana I. The role of the eyes and the pineal

Summary The pineal and the eyes are known to be important components in the circadian system of some species of lizards; their effects may be mediated by the hormone melatonin. We examined the role played by these structures in the desert iguana (Dipsosaurus dorsalis). Surgical removal of the pineal had no effect on circadian locomotor rhythms, even though this procedure abolished the circadian rhythm of melatonin in the blood. Furthermore, when the isolated pineal of Dipsosaurus was studied in organ culture, it showed no circadian rhythm of melatonin secretion, as do pineals of some other lizard species, although it did produce large quantities of this hormone. Bilateral ocular enucleation had only small effects on the freerunning period of locomotor rhythms, without affecting melatonin levels in the blood. Behavioral circadian rhythms persisted in desert iguanas subjected to both enucleation and pinealectomy. These data suggest that neither the pineal nor the eyes are central components of the circadian pacemaking system in Dipsosaurus, nor is melatonin critically involved in maintaining its organization.Abbreviations CT circadian time - ZT zeitgeber time - LL constant light - LD light-dark cycle - DD constant darkness - freerunning circadian period