Membrane associated events during proliferative inhibition of granulocyte precursors

A new way of assessing the significance of intracellular signals that may regulate cellular proliferation, would be to analyze possible 'second messengers' when proliferation is slowed down, rather than stimulated. Therefore, we examined proliferating mononuclear blood cells from leukaemic patients which had been exposed to an inhibitory ox leucocyte extract. The extract decreased 3H-thymidine incorporation in leukaemic cells in short-term cultures. The inhibition was not cell-line specific, but was nevertheless non-toxic and not due to endotoxin. The K+ flux into the leukaemic cells was assessed with 86Rb+, a K+ analogue. An inverse relationship was found between 86Rb+ uptake and 3H-thymidine incorporation. The increased 86Rb+ influx was probably due to leakage or exchange mechanisms other than the Na+/K+ membrane pump, as suggested by ouabain inhibition experiments. However, the long lag time (greater than 45 min) between addition of inhibitor and a marked increase in 86Rb+ uptake does not support a role for the K+ flux as an early mediator of the inhibitory signal.     

Production of proliferation inhibitors by mature granulocytes

The aim of the experiments was to find ways of increasing the yield of small molecular weight inhibitors of cell proliferation released by granulocytes. Almost pure populations of granulocytes from pig or human blood, or from sterile inflammatory exudates in rats were treated in various ways and then spun down. Molecules below approximately 10 000 dalton (Diaflo ultrafiltration or Sephadex G 25 filtration) in the supernatants were tested for inhibitory activity by measuring 3H-thymidine incorporation in 5 to 6-h coverslip cultures of rat bone marrow cells. The different granulocyte treatments were: Freeze-thawing, sonication, incubation (at +4 degrees -37 degrees C) in hypotonic media (0-200 mosm/kg), storage in vitro overnight (at +4 degrees C) before incubation, incubation at 37 degrees C in complete and buffered tissue culture medium (Fischer's with 10 mmol/1 HEPES), incubation in saline only (2-h periods, approximately 70 X 10(6) cells/ml), or with lidocaine added, with Ca++ and the Ca++ ionophore A-23187, with K+ and the K+ ionophore Valinomycin, with a high K+ concentration (50 mmol/1), with arachidonic acid, with a cAMP analogue, or with a protease inhibitor added during or at the end of the incubation. On a per cell basis rat peritonitis granulocytes released more inhibitor than pig blood granulocytes, whereas human blood granulocytes were not detectably inhibitory at all. Arachidonic acid was the most promising agent tested to increase inhibitor release above that occurring spontaneously from granulocytes incubated in saline.     

Morphologische untersuchungen an zwei drüsentypen (Thoraxdrüse,Mandibeldrüse) von Lepidopterenraupen

Two types of possibly homologous glands different in structure and formation have been found. One of them is represented in C. vinula L. and N. anceps Goeze, it is an endocrine organ. The other in S. ligustri, S. ocellata, M. neustria and L. monacha has an excretory duct and therefore is an exocrine organ.

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Mit dankenswerter Unterstützung der Deutschen Forschungsgemeinschaft.     

Characterization of the Rhizobium leguminosarum biovar phaseoli nifA gene,a positive regulator of nif gene expression

We report the isolation, mutational analysis and the nucleotide sequence of the Rhizobium leguminosarum bv. phaseoli nifA gene. Comparison of the deduced amino acid sequence with other NifA sequences indicated the presence of the conserved central activator and the C-terminal DNA-binding domains. Nodules elicited by a R. leguminosarum bv. phaseoli nifA mutant were symbiotically ineffective. The expression of a nifA-gusA fusion was shown to be independent on the oxygen status of the cell. We cloned the three nifH copies of R. leguminosarum bv. phaseoli and determined the nucleotide sequence of their promoter regions. The expression of nifH-gusA fusions is induced under microaerobic conditions and is dependent on the presence of NifA.Abbreviations bp base pair(s) - kb kilobase(s) - ORF open reading frame     

Agitation,aeration and perfusion modules for cell culture bioreactors

For an optimized bioreactor design which is adapted to the cultivation of sensitive animal cells different modular bioreactor components for gentle agitation, sufficient aeration and long-term perfusion were developed and investigated with respect to their suitability from laboratory to production scale. Aeration systems have been designed for both shear sensitive cells and cells which tolerate bubbles. The systems are based on either membranes for bubble-free aeration or stainless steel sparger systems. They were characterized by determination of their oxygen transfer capacity and optimized in cultivation processes of different cell lines under process conditions such as batch and perfusion mode.Different impellers for suspension cells and cells grown on carriers were investigated for their suitability to ensure homogeneous gentle mixing. A large pitch blade impeller as well as a novel 3-blade segment impeller are appropriate for homogeneous mixing at low shear rates. Especially with the 3-blade segment impeller fluid mechanical stress can be reduced at a given stirrer speed which is advantageous for the cultivation of cells attached to microcarriers or extremely shear sensitive suspension cells. However, our results indicate that shear sensitivity of animal cells has been generally overestimated.Continuous perfusion of both suspension cell cultures and cells cultivated on microcarriers could be successfully performed over extended periods of time using stainless steel spinfilters with appropriate pore sizes and systems based on microporous hydrophilic membranes. Spinfilters are suitable cell retention systems for technical scale bioreactors allowing continuous perfusion cultures of suspension cells (pore size 10 to 20 m) as well as anchorage dependent cells grown on microcarriers (pore size 75 m) over six weeks to 3 months.Applying the developed modules for agitation, aeration and perfusion process adapted bioreactor set-ups can be realized which ensure optimum growth and product formation conditions in order to maximize cell and product yields.     

The ultrastructure of campaniform sensilla on the eye of the cricket,Gryllus campestris

Summary The structure of the campaniform sensilla of the cricket eye was investigated by light and electron microscopy. Each sensillum is innervated by a single bipolar neuron. Its axon extends through the retina into a side-branch of the nervus tegumentarius. The dendrite extends through a cuticular channel to the surface of the cornea. The distal part of the dendrite, the sensory process, contains a tubular body and is attached to a cuticular cap which is obliquely inserted into the exocuticle between the corneal lenslets. Some particular structural features as well as the function of the campaniform sensillum of the cricket eye are discussed.Supported by the Deutsche Forschungsgemeinschaft, grant Ho 463/10The authors are indebted to Prof. H. Altner, University of Regensburg, and Mrs. Evelyn Thury, Contron GmbH, München for use of the scanning electron microscope facilities     

UPTAKE OF GLUCOSE ANALOGUES BY RAT BRAIN CORTEX SLICES: Na+-INDEPENDENT MEMBRANE TRANSPORT

Abstract— The glucose analogues 3-O-methyl-D-glucose and α-methyl-D-glucoside were not metabolized in brain tissue.
The uptake of these two sugars into the intracellular compartment of brain cortex slices was investigated using media with normal and low Na⁺ concentration (replacement of all NaCl with choline Cl). The cellular transport was not Na⁺-dependent. The transport mechanism clearly distinguished between the two sugars in both normal and low Na⁺ media.     

Veränderungen der Oenozyten und ihre Funktion während der Metamorphose bei Schwärmern und Zahnspinnern

Zusammenfassung VonCerura vinula undSphinx ligustri wurden die Oenozyten im letzten Larvenstadium und in der Puppe untersucht und ihre Struktur beschrieben. Ihre Aktivitätsphasen liegen zur Zeit der beiden Häutungen (Larven- und Puppenhäutung) und im letzten Larveninstar vor der Umfärbung, einem äußerlich sichtbaren Metamorphoseschritt, und vor der Puppenhäutung im Färbungsstadium III. Sie stehen mit den Umwandlungsprozessen, die in den Raupen zu diesem Zeitpunkt stattfinden in deutlichem Zusammenhang. —2–4 Monate nach der Puppenhäutung sind in den diapausierenden Puppen noch aktive larvale Oenozyten vorhanden. — Aktivitätsphasen sind charakterisiert durch viele große und kleine Vakuolen neben kanalartigen Strukturen im Zytoplasma, stark verzweigte Kerne und weitreichende Zellaus- und-einbuchtungen.Im Vorpuppenstadium (Färbungsstadium IV) entstehen die imaginalen Oenozyten aus der Epidermis, sie werden erst kurz vor der Adulthäutung aktiv.Haemozyten, neurosekrethaltig, legen sich dicht an die Oenozyten an und dringen zwischen Zelleinfaltungen ins Innere vor.Lipide, besonders reichlich in aktiven Phasen vorhanden, konnten sowohl im Zytoplasma nachgewiesen werden, als auch ihr Übertritt aus dem Fettgewebe, das den Drüsen eng anliegt.Glykogen tritt ebenfalls in den Oenozyten auf, seine Menge steht aber in keinem merklichen. Zusammenhang mit den Zellrhythmen. Physiologische Versuche beweisen, daß die Oenozyten und auch die Prothorakaldrüsen in aktiven Phasen das Häutungs- und Metamorphosehormon abgeben. Sie lösen beide den Umfärbungsprozeß aus. Gehirne mit neurosekretorischen Zellen in aktiver Phase oder Cholesterin können die Prothorakaldrüsen und z.T. auch die Oenozyten zur Abgabe ihres Hormons anregen.

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Changes of oenocytes and their function during metamorphosis of sphingidae and notodontidae

Summary Oenocytes of the last larval instar and the pupa ofCerura vinula andSpinx ligustri have been examined, and their structure described. The activity phases of the oenocytes at the time of both moultings, as well as during the last larval instar prior to an externally visible color change and prior to the pupal ecdysis i.e. during color change stage III) were clearly related to the process of metamorphosis, which was occurring in the larvae at this time 2–4 months after pupal ecdysis, diapausing pupae still show active larval oenocytes. Activity phases are characterized by many large and small vacuoles in addition to channel-like cytoplasmatic structures, heavily branched nuclei and extensive cell processes and infoldings of the cell membrane. In the pharate pupal stage (colour change stage IV) the imaginal oenocytes originate from the hypodermis, becoming active just prior to the adult ecdysis.Haemocytes containing neurosecretory material attach themselves to the oenocytes and enter through infoldings of the cell membrane. Lipids, which are particularly abundant during active phases, could be demonstrated in the cytoplasm as well as passing from the fatty tissue closely surrounding the glands. Glycogen was also present in the oenocytes. There was, however, no noticeable relation of these materials to the rhythm.Physiological experiments demonstrated that oenocytes as well as prothoracic glands, when active, secrete the moulting and metamorphosis hormone. Both glands initiate the process of colour change. Brain tissue, containing active neurosecretory cells, or cholesterol, may stimulate the prothoracic galnds and the oenocytes to secrete their hormone.

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Mit dankenswerter Unterstützung durch die Deutsche Forschungsgemeinschaft.