Studies of protoplast fusion in Streptomyces chrysomallus

Conditions for the regeneration of cells from protoplasts of Streptomyces chrysomallus, a producer of the peptide antibiotic actinomycin, are described. Regeneration of fusion products was most efficient at 27-30 degrees C on regeneration R2 medium (Okanishi et al., 1974) containing 0.25 M-sucrose. The addition of phosphate (150-300 mg 1(-1) to the medium and incubation at 23 degrees C proved to be optimal for the regeneration of individual strains. Highest recombination frequencies after protoplast fusion were obtained by fusing protoplasts in the presence of 45% (w/v) polyethylene glycol 6000. With strains that produce no, or little antibiotic, protoplasts must be present in excess in fusion mixtures in order to overcome inhibition of regeneration by the antibiotic-producing partner.     

Mechanism of depsipeptide formation catalyzed by enniatin synthetase

Covalently bound intermediates of enniatin B synthesis could be isolated from enniatin synthetase by treatment with performic acid. By comparison with products of mild alkaline cleavage of authentic enniatin B they could be identified as the dipeptide D-2-hydroxyisovaleryl-N-methylvaline and the corresponding tetrapeptide. Synthesis of enniatins apparently proceeds via condensation of dipeptides. This was confirmed by the use of the substrate analogue isovaleric acid, which has shown to be a strong inhibitor for enniatin synthesis by formation of N-isovaleryl-N-methyl valine.     

Restricted pH ranges and reduced yields for bacterial growth under pressure

Hydrostatic pressure was found to cause a marked narrowing of pH ranges for growth and reductions in growth yields for a variety of bacteria. In many cases, reduced yields under pressure could be directly related to increased sensitivities to metabolic acids that accumulated in the enclosed culture vessels used. Magnesium and calcium ions partially reversed increases in sensitivities of representative gram-positive and gram-negative bacteria to low, but not high, pH. Growth inhibition of these organisms at both extremes of pH was associated with enhanced loss of K⁺ from pressurized cells. Inhibited cells in alkaline media also lysed under pressure, but microscopically observable lysis was clearly a secondary phenomenon because it occurred slowly. Apparent volumes for growth-inhibitory protonation-deprotonation reactions were calculated on the basis of measured shifts in inhibitory pH with pressure. The values ranged from 99 to 431 ml/mole, and their magnitudes indicated that growth inhibition by acids or bases involves cooperative changes in polymeric interactions such as those which accompany protein denaturation.     

Zur Feinstruktur von Cuticula und Epidermis beim Flußkrebs Orconectes limosus während eines Häutungszyklus

Zusammenfassung Die Cuticula an der Innen- und Außenseite der Branchiostegite des Flußkrebses besteht wie für Arthropoden üblich aus Epi- und Procuticula. Sowohl die Epicuticula als auch die Procuticula von Innen- und Außenseite unterscheiden sich im Feinbau wesentlich voneinander. An der Innenseite ist die Epicuticula einfach gebaut; Die Procuticula ist lamelliert und zeigt meist die bogenförmigen Muster von Mikrofibrillen. Die Epicuticula an der Außenseite weist in den in dieser Arbeit untersuchten Entwicklungsstadien einen sehr viel komplizierteren Feinbau auf, der in der Entwicklung gewissen Änderungen unterliegt. In der wiederum lamellierten Procuticula an der Außenseite sind die Mikrofibrillen zu Balken gebündelt. Die Ausrichtung der Mikrofibrillen dreht sich innerhalb einer Lamelle um 180°. Durch die Procuticula ziehen Fortsätze der Epidermiszellen, außerdem Stäbe der sog. Verbindungsstrukturen.Die Bildung der Cuticula an der Innenseite konnte weitgehend vollständig verfolgt werden; sie ist gut mit der Bildung der Cuticula bei verschiedenen Insekten vergleichbar.Die Bildung der Cuticula an der Außenseite konnte dagegen nur von Beginn der Abscheidung der Proouticula bis zur Häutung verfolgt werden. Kurz vor Beginn der Cuticulaabscheidung kommt es in den Epidermiszellen zu einer stärkeren Entwicklung des rauhen ER. Während der gesamten von uns verfolgten Bildungsstadien sieht man Vesikel mit dichtem Inhalt besonders in der Nähe des Zellapex. Sie geben anscheinend hier ihren Inhalt, Cuticulamaterial, nach außen ab. Sie stammen wohl aus Golgibereichen. Auch Stachelsaumbläschen (coated vesicles) kommen regelmäßig vor, deren genetischer Zusammenhang mit multivesikulären Körpern diskutiert wird. Bei der Abscheidung der fibrillären Cuticulasubstanzen spielen besondere Differenzierungen der Zell oberfläche, — kappenartige Verdichtungen der Zelloberfläche, meist an der Spitze kleiner Mikrovilli — eine wesentliche Rolle.

---

The ultrastructure of cuticle and epidermis in the crayfish Crconectes limosus during a moulting cycle

Summary The cuticle of the inside and outside of the branchiostegites of the crayfish consists of an epicuticle and a procuticle — as common in arthropods. Concerning their ultrastructure epicuticle and procuticle differ essentially from each other on both the inside as well as the outside. On the inside the epicuticle is built plainly; the procuticle is laminated, and, mostly it shows the arched patterns of microfibrils. In those developmental stages investigated in this project the epicuticle of the outside shows a much more intricated ultrastructure, since during formation it is subject to certain changes. On the outside the procuticle is also laminated; the microfibrils are bundled up to bars. The alignment of those microfibrils within one lamella is twisted for 180°. The procuticle is penetrated by processes of epidermal cells and by rods of the so-called connecting structures.The formation of the cuticle on the inside was observed completely; it is comparable to the forming of the cuticle in several insects. However, the formation of the cuticle on the outside was only observed from the beginning of the procuticular development up to the moulting.Shortly before formation of the cuticle the development of rough ER in the epidermal cells seems to be intensified. In all of developmental stages observed there appear vesicles with dense contents mainly situated nearby the cell apex. At this site they evidently deliver their contents — cuticular materials — to the outside of the cell; they probably originate in the Golgi areas. There occur coated vesicles regularity, too; their genetic relation to multivesicular bodies is discussed. Special differentiation on the cell surface i.e. dome-like consolidations of the cell surface mainly placed at the tip of small microvilli are of great importance for the secretion of the cuticle substances.

     

The effect of iron chelate fertilization of poplar upon CO2-uptake,leaf size,and content of leaf pigments and iron

Summary Potted poplars (strainsmarilandica, serotina andFlachslanden ofPopulus euramericana) which developed iron-deficiency symptoms (chlorosis of upper leaves, winter die-back of leader, flushing of lateral buds) were treated with a soil application of iron chelate to study the effect of iron nutrition upon CO₂-uptake, iron and pigment content of leaves, and leaf size of a tree species. Foliar content of each iron, chlorophyll, -carotene, lutein, and violaxanthin was significantly increased by the treatment. Chlorophyll b proved to be particularly sensitive to iron supply and the Q^a/b was also significantly altered.CO₂-uptake increased in fertilized and non-fertilized leaves with increasing light up to 40,000 Lux, but fertilized leaves assimilated more CO₂ than non-fertilized leaves, especially at light intensities from 5,000 Lux upwards. The assimilatory number was decreased by the iron application since larger amounts of chlorophyll were present in fertilized leaves. If CO₂-uptake was based upon an area unit basis the fertilizer effect became distinct even at 500 Lux. Thus CO₂-uptake is a quick, valuable measure of fertilizer responses.In severe cases, iron deficiency also affects leaf size and thus indirectly reduces photosynthetic activity. A chelate application during the growing season will not affect the size of leaves already formed but may considerably increase the size of leaves formed subsequent to the treatment.     

Induction of microspore embryogenesis inBrassica napus L. by gamma irradiation and ethanol stress

Summary Gamma irradiation and ethanol stress treatments redirected pollen development to an embryo formation pathway inBrassica napus. Less than 0.01% of microspores developed into embryos at 25°C compared to approximately 2% at 32°C. However, subsequent to gamma irradiation and ethanol treatments up to 1% and 0.7% of microspores formed embryos at 25°C, respectively. Gamma irradiation also enhanced embryogenesis at 32°C. The possible importance of these findings is discussed in relation to microspore embryogenesis.     

Respiratory sinus arrhythmia in the denervated human heart

We performed this study to test whether the denervated human heart has the ability to manifest respiratory sinus arrhythmia (RSA). With the use of a highly sensitive spectral analysis technique (cross correlation) to define beat-to-beat coupling between respiratory frequency and heart rate period (R-R) and hence RSA, we compared the effects of patterned breathing at defined respiratory frequency and tidal volumes (VT), Valsalva and Mueller maneuvers, single deep breaths, and unpatterned spontaneous breathing on RSA in 12 normal volunteers and 8 cardiac allograft transplant recipients. In normal subjects R-R changes closely followed changes in respiratory frequency (P less than 0.001) but were little affected by changes in VT. On the R-R spectrum, an oscillation peak synchronous with respiration was found in heart transplant patients. However, the average magnitude of the respiration-related oscillations was 1.7-7.9% that seen in normal subjects and was proportionally more influenced by changes in VT. Changes in R-R induced by Valsalva and Mueller maneuvers were 3.8 and 4.9% of those seen in normal subjects, respectively, whereas changes in R-R induced by single deep breaths were 14.3% of those seen in normal subjects. The magnitude of RSA was not related to time since the heart transplantation, neither was it related to patient age or sex. Thus the heart has the intrinsic ability to vary heart rate in synchrony with ventilation, consistent with the hypothesis that changes, or rate of changes, in myocardial wall stretch might alter intrinsic heart rate independent of autonomic tone.     

Transforming growth factor-beta 1 enhances the suppression of human hematopoiesis by tumor necrosis factor-alpha or recombinant interferon-alpha

The effects of transforming growth factor-beta 1 (TGF-beta 1) on human hematopoiesis were evaluated in combination with two other regulatory cytokines, namely, recombinant human tumor necrosis factor-alpha (TNF-alpha) and recombinant human interferon-alpha (rIFN-alpha). Combinations of TNF-alpha and TGF-beta 1 resulted in a synergistic suppression of colony formation by erythroid progenitor cells (BFU-E) and an additive suppression of granulocyte-macrophage (CFU-GM) and multipotential (CFU-GEMM) progenitor cells. In addition, TGF-beta 1 synergized with rIFN-alpha to suppress CFU-GM formation, while the combined suppressive effects of both cytokines on CFU-GEMM and BFU-E were additive. When TGF-beta 1 was tested with TNF-alpha or IFN-alpha on granulocyte/macrophage colony-stimulating factor (GM-CSF)-stimulated bone marrow cells in a 5-day proliferation assay, the antiproliferative effects of TGF-beta 1 and TNF-alpha were additive, while those with TGF-beta 1 and rIFN-alpha were synergistic. A similar pattern was seen in the suppression of the myeloblastic cell line KG-1 where TGF-beta 1 in combination with TNF-alpha resulted in an additive suppression while inhibition by TGF-beta 1 and IFN-alpha was synergistic. These results demonstrate for the first time the cooperative effects between TGF-beta and TNF-alpha and IFN-alpha in the suppression of hematopoietic cell growth, raising the possibility that TGF-beta might be used in concert with TNF-alpha or IFN-alpha in the treatment of various myeloproliferative disorders.