Application of an Escherichia coli triple reporter strain for at-line monitoring of single-cell physiology during L-phenylalanine production

Biotechnological production processes are sustainable approaches for the production of biobased components such as amino acids for food and feed industry. Scale-up from ideal lab-scale bioreactors to large-scale processes is often accompanied by loss in productivity. This may be related to population heterogeneities of cells originating from isogenic cultures that arise due to dynamic non-ideal conditions in the bioreactor. To better understand this phenomenon, deeper insights into single-cell physiologies in bioprocesses are mandatory before scale-up. Here, a triple reporter strain (3RP) was developed by chromosomally integrating the fluorescent proteins mEmerald, CyOFP1, and mTagBFP2 into the L-phenylalanine producing Escherichia coli strain FUS4 (pF81_kan) to allow monitoring of growth, oxygen availability, and general stress response of the single cells. Functionality of the 3RP was confirmed in well-mixed lab-scale fed-batch processes with glycerol as carbon source in comparison to the strain without fluorescent proteins, leading to no difference in process performance. Fluorescence levels could successfully reflect the course of related process state variables, revealed population heterogeneities during the transition between different process phases and potentially subpopulations that exhibit superior process performance. Furthermore, indications were found for noise in gene expression as regulation strategy against environmental perturbation.     

Tumor necrosis factors in 1996

The 6th International Congress on Tumor Necrosis Factors and Related Molecules was held in Faliraki, Island of Rhodes, Greece, 8–12 May, 1996. This review summarizes the topics addressed and highlights some of the major advances presented during the meeting.     

Global vegetation models: incorporating transient changes to structure and composition

Abstract. We describe an approach for developing a Dynamic Global Vegetation Model (DGVM) that accounts for transient changes in vegetation distribution over a decadal time scale. The DGVM structure is based on a linkage between an equilibrium global vegetation model and smaller scale ecosystem dynamics modules that simulate the rate of vegetation change. Vegetation change is classified into four basic types, based largely on the projected change in above-ground biomass of the vegetation. These four types of change are: (1) dieback of forest, shrubland or grassland; (2) successional replacement within forest, shrubland or grassland; (3) invasion of forest, shrubland or grassland; (4) change in tree/grass ratio. We then propose an approach in which the appropriate ecosystem dynamics module for each type of change is applied and the grid cells of the global model updated accordingly. An approach for accounting for fire, as an example of a disturbance which may strongly influence the rate and spatial pattern of forest dieback, is incorporated. We also discuss data needs for the development, calibration and validation of the model.     

Genomic structure and chromosomal location of the mouse pre-T-cell receptor alpha gene

The mouse pre-T-cell receptor alpha (pT) chain is a 33 000 M _r glycoprotein expressed on the surface of immature thymocytes as a disulfide-linked heterodimer with the T-cell receptor beta (TCR) chain, and in association with proteins of the CD3 complex. The cDNA for pT, isolated previously, encodes a type I transmembrane protein that is a member of the immunoglobulin (Ig) superfamily. Here we report the complete nucleotide sequence, the exon/intron structure, and the chromosomal location of the pTa gene. The gene spans about 8.4 kilobases (kb) and consists of four exons. Exon 1 encodes the 5 untranslated region, the leader peptide, and the first three amino acids of the mature protein. This exon is followed by a relatively long intron of 4.9 kb that contains many short interspersed repeats (SINEs) of the B1 and B2 family. The second exon encodes the extracellular Ig-like domain and exon 3 with just 45 base pairs the connecting peptide (CP), including the cysteine required for heterodimer formation. A similar exon/intron structure encoding corresponding parts of the mature polypeptide is found both in the Tcra and Tcrd constant region genes. The last exon encodes the transmembrane portion, the cytoplasmic tail, and about 540 nucleotides of 3 untranslated sequence, including a B2 repetitive element. In situ hybridization maps the pTa gene to the D/E1 region of mouse chromosome 17.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number U27268     

Effect of cell cycle on the regulation of the cell surface and secreted forms of type I and type II human tumor necrosis factor receptors

The cell cycle has been shown to regulate the biological effects of human tumor necrosis factor (TNF), but to what extent that regulation is due to the modulation of TNF receptors is not clear. In the present report we investigated the effect of the cell cycle on the expression of surface and soluble TNF receptors in human histiocytic lymphoma U-937. Exposure to hydroxyurea, thymidine, etoposide, bisbensimide, and democolcine lead to accumulation of cells primarily in G₁/S, S, S/G₂/M, G₂/M, and M stages of the cell cycle, respectively. Whilie no significant change in TNF receptors occurred in cells arrested in G₁/S or S/G₂ stages, about a 50% decrease was observed in cells at M phase of the cycle. Scatchard analysis showed a reduction in receptor number rather than affinity. In contrast, cells arrested at S phase (thymidine) showed an 80% increase in receptor number. The decrease in the TNF receptors was not due to changes in cell size or protein synthesis. The increase in receptors, however, correlated with an increase in total protein synthesis (to 3.8-fold of the control levels). A proportional change was observed in the p60 and p80 forms of the TNF receptors. A decrease in the surface receptors in cells arrested in M phase correlated with an increase in the amount of soluble receptors. The cellular response to TNF increased to 8- and 2-fold in cells arrested in G₁ and S phase, respectively; but cells at G2/M phase showed about 6-fold decrease in response. In conclusion, our results demonstrate that the cell cycle plays an important role in regulation of cell-surface and soluble TNF receptors and also in the modulation of cellular response. © 1995 Wiley-Liss, Inc.     

Immunocytochemical colocalization of adhesive proteins with clathrin in human blood platelets: further evidence for coated vesicle-mediated transport of von Willebrand factor,fibrinogen and fibronectin

Coated membranes and vesicles play an important role in receptor-mediated endocytosis and intracellular trafficking in various cell types, and are also present in blood platelets. Platelets take up certain proteins from the blood plasma, such as von Willebrand factor and fibrinogen, and these substances are transferred to storage granules. The receptors for these plasma proteins on the platelet plasma membrane have been well characterized, but morphological evidence for their transport to the storage granules is not yet available. In an attempt to clarify this aspect, we employed postembedding immunocytochemistry on platelets embedded in the acrylic resin LR White. Clathrin as the major coat component of coated vesicles was localized in the cytoplasm, on the plasmic faces of -granules and the open canalicular system, and on the plasmic face of the plasma membrane. Colocalizations of the adhesive proteins, von Willebrand factor, fibrinogen and fibronectin, with clathrin could be observed at the same typical locations as coated vesicles were seen in Araldite-embedded material. These colocalizations have not been reported to date and furnish further evidence for a coated vesicle-mediated transport of blood plasma-derived adhesive proteins from their receptors on the outer plasma membrane to the -granules.     

Die postembryonale Entwicklung und die funktionelle Anatomie des Gnathosoma in der Milbenfamilie Erythraeidae (Acarina,Prostigmata)

Zusammenfassung Das Gnathosoma von sieben Arten der Milbenfamilie Erythraeidae wurde untersucht. Die postembryonale Entwicklung der Cuticula-Strukturen, der Muskulatur und der Drüsen wird beschrieben.Das Gnathosoma der Larven weist Cheliceren auf, die aus Grundglied und klauenförmigem Digitus mobilis bestehen. Sie liegen dem Infracapitulum dorsal auf und inserieren an einem Paar Capitular-Apophysen. Bei den Protound Tritonymphen unterliegt das Gnathosoma der Histolyse und wird anschließend jeweils neu ausgebildet. Bei der Deutonymphe und beim Adultus ist es gegenüber der Larve stark abgewandelt: Die Tracheenstämme sind aus den Capitular-Apophysen der Larve abzuleiten. Die Stech-Cheliceren entsprechen dem Grundglied der larvalen Cheliceren, und ihre medialen Protraktoren gehen aus den Levator-Muskeln der Cheliceren der Larve hervor.Der Weg der Sekrete der podocephalischen und infracapitulären Drüsen über die Dorsalfläche des Infracapitulum erfährt mit dem Erwerb schmaler Stech-Cheliceren eine radikale Umstellung des Schutzes gegen Austrocknung. Während bei der Larve die breiten Cheliceren-Grundglieder den Sekretweg überdecken, und der Raum zwischen den Grundgliedern und dem Infracapi-tulum zusätzlich durch das ölartige Sekret der Intercheliceraldrüse ausgefüllt wird, schließen sich bei der Deutonymphe und beim Adultus die Lateralkiele der Genae über den Stech-Cheliceren zusammen, und der hintere Abschnitt des Cervix wird in das Idiosoma eingesenkt. Dadurch wird der Cervix zu einem internen Cervicalkanal umgebildet.Der Mundraum wird bei den aktiven Stadien durch die lipidartigen Sekrete der Buccal- und Labialdrüsen geschützt.Die phylogenetischen Beziehungen der Parasitengona innerhalb der Prostigmata und der Erythraeidae innerhalb der Parasitengona werden diskutiert.

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Postembryonic development and functional anatomy of the gnathosoma in the family Erythraeidae (Acarina, Prostigmata)

Summary The gnathosoma of seven species of the Erythraeidae was investigated. The postembryonic development of the cuticular structures, the musculature and the glands are described.In the larval gnathosoma, the chelicerae consist of a basal segment and a claw. They rest upon the dorsal surface of the infracapitulum. The basal segments are attached to a pair of capitular apophyses (sigmoid pieces). During the two molts larva to protonymph and deutonymph to tritonymph, the gnathosoma is histolysed. Directly afterwards it is rebuilt. Compared to the larva, in the deutonymph and in the adult it undergoes profound changes: The capitular apophyses are transformed to parts of the tracheae, the basal segment of each chelicera to a styliform chelicera, and the levator muscles of the chelicerae to medial protractors of the styliform chelicerae.The secretions of the podocephalic and infracapitular glands proceed along the dorsal surface of the infracapitulum to the buccal cavity. In the larva, the way of the secretions is protected against desiccation by the broad basal segments of the chelicerae, that cover the infracapitulum. In addition the oily secretion of the intercheliceral gland seals the space between the infracapitulum and the chelicerae. By obtaining styliform chelicerae the protection against desiccation undergoes a radical change: The lateral ridges of the infracapitulum join above the chelicerae, and the posterior part of the cervix is transferred back into the idiosoma. Thus the cervix is transformed into an internal canal.In the active instars, the buccal cavity is protected by the lipid-like secretions of the buccal glands and labial glands.The phylogenetic relationships of the Parasitengona within the Prostigmata, and of the Erythraeidae within the Parasitengona are discussed.