Cytochemical localization of β-galactosidase in resident and inflammatory peritoneal macrophages from C57BL mice

Summary A cytochemical method for the detection of -galactosidase (-Gase) in mouse peritoneal macrophages was used to study the ultrastructural localization of this enzyme in these cells. It was found that the reaction product for -Gase was localized in the perinuclear cisternae, the endoplasmic reticulum, the Golgi complex, lysosomes, vesicles and on the cell surface of peritoneal macrophages from untreated C57BL mice. When examined by X-ray microanalysis the crystalline reaction product was found to contain bromine, an element present in the indolyl substrate which was used to identify -Gase. Injection of Proprionibacterium acnes (P. acnes) intraperitoneally or BCG intravenously caused a visible loss in -Gase from all the organelles and from the cell surface of the macrophages.Abbreviations used -Gase -galactosidase - RP reaction product - PNC perinuclear cisternae - RER rough endoplasmic reticulum     

Predation and potential transfer of pollen in a population of Saxifraga hirculus

Body surface pollen load and gut pollen (pollen diet load) of flower visitors in a Danish population of Saxifraga hirculus were analyzed. The four most frequent visitors, Eurimyia lineata (Syrphidae), Neoascia tenur (Syrphidae), Asindulum nigrum (Mycetophilidae), and Zygaena trifolii (Zygaenidae) had total surface loads (and S. hirculus loads) of 1031 (771), 137 (106), 108 (78), and 318 (27) pollen grains, respectively. Other syrphid visitors had on average a load of 407 (192) pollen grains. Males of E. lineata, N. tenur , and A. nigrum carried more surface pollen than the females; females of E. lineata had a lot of pollen from Ranunculus acris and S. hirculus and males had a lot from S. hirculus . Females of N. tenur carried pollen mostly from S. hirculus, Lotus uliginosus and Galium uliginosum ; males carried pollen mostly from S. hirculus . The surface pollen load of A. nigrum was dominated by S. hirculus pollen, whereas that of Z. trifolii was dominated by Cirsium palustre and Lychnis flos-cuculi pollen. The amount and kind of pollen varied enormously both intraspecifically and interspecifically. Eurimyia lineata, N. tenur and other syrphids had total pollen diet loads (and S. hirculus pollen diet loads) of approximately 14900 (657), 3739 (2327) and 17784 (13040). The average syrphid contained pollen from five plant species, only one to two, however, were quantitatively important. The average diet load in females was nine times that in males in both E. lineata and N. tenur. Eurimyia lineata ate a lot of R. acris; N. tenur ate a lot of S. hirculus and G. uliginosum . Generally, no correlation was observed between size or season and the composition of surface load and diet load. Eurimyia lineata made most of the pollen visits. Thus, the seed set from this Danish population of S. hirculus was dependent on a single syrphid species.     

The contrasting role of auxin in submergence-induced petiole elongation in two species from frequently flooded wetlands

The involvement of auxin in the submergence-induced petiole elongation has been investigated in Rumex palustris and Ranunculus sceleratus. Both wetland species are capable of enhanced petiole elongation upon submergence or treatment with exogenous ethylene (5μl l⁻¹). Treatment of intact Rumex palustris plants with 1-naphthalene acetic acid (NAA) at 10⁻⁴ M enhanced petiole elongation, while treatment with N -1-naphthylphthalamic acid (NPA) had no effect on petiole elongation. The elongation response after NAA or NPA treatment was comparable for plants in both submerged and drained conditions. Pre-ageing of detached petioles of Rumex palustris for 3 h in light or in dark conditions had no effect on the submergence-induced elongation. In comparison to intact plants, detached petioles of Rumex palustris , with or without lamina, did not show significant differences in responsiveness to IAA between drained or submerged conditions. This was in contrast to Ranunculus sceleratus where submergence caused a clear increase in responsiveness towards IAA. Removal of the lamina, the putative source of auxin, or treatment with NPA did not hinder the submergence-induced elongation of detached Rumex palustris petioles, but severely inhibited elongation of detached Ranunculus sceleratus petioles. This inhibition could be restored by application of NAA, suggesting the specific involvement of auxin in the submergence response of Ranunculus sceleratus. It is concluded that, in contrast to Ranunculus sceleratus , auxin is probably not involved in the submergence-induced petiole elongation of Rumex palustris.     

Potamogeton pectinatus Is Constitutively Incapable of Synthesizing Ethylene and Lacks 1-Aminocyclopropane-1-Carboxylic Acid Oxidase

A highly sensitive laser-driven photoacoustic detector responsive to [less than or equal to]2.1 nmol m-3 ethylene (50 parts per trillion [v/v]) was used for ethylene analysis. Dark-grown plants of Potamogeton pectinatus L. growing from small tubers made no ethylene. Exposure of shoots to white light, wounding, submergence in water followed by desubmergence, partial oxygen shortage, indole acetic acid, or carbon dioxide failed to induce ethylene production, although clear effects were observed in Pisum sativum L. Some ethylene was released after applying high concentrations of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC; 10 mol m-3) to P. pectinatus, but the amount was trivial compared with that released by P. sativum. More endogenous ACC was found in P. pectinatus than in P. sativum. Considerable ACC oxidase activity was present in tissue extracts of P. sativum. However, no ACC oxidase activity was found in P. pectinatus, indicating that this is where ethylene production is arrested.     

Localization and organization of phenol degradation genes ofPseudomonas putida strain H

The genetic organization of the DNA region encoding the phenol degradation pathway ofPseudomonas putida H has been investigated. This strain can utilize phenol or some of its methylated derivatives as its sole source of carbon and energy. The first step in this process is the conversion of phenol into catechol. Catechol is then further metabolized via themeta-cleavage pathway into TCA cycle intermediates. Genes encoding these enzymes are clustered on the plasmid pPGH1. A region of contiguous DNA spanning about 16 kb contains all of the genetic information necessary for inducible phenol degradation. The analysis of mutants generated by insertion of transposons and cassettes indicates that all of the catabolic genes are contained in a single operon. This codes for a multicomponent phenol hydroxylase andmeta-cleavage pathway enzymes. Catabolic genes are subject to positive control by the gene product(s) of a second locus.     

Evaluation of mass spectrometric techniques for charaterization of engineered proteins

A simple and versatile method of in vitro site-specific mutagenesis based on polymerase chain reaction (PCR) is described. The complete method required the use of three oligonucleotide primers and two PCRs. The product of the first PCR was used as one of the primers (megaprimer) in the second PCR. Essentially 100% of the final product incorporated the desired mutation. The various aspects of the procedure and its application is described in detail.     

Localisation of sulfakinin neuronal pathways in the blowfly Calliphora vomitoria

The distribution of neurones immunoreactive to antisera raised against the undecapeptide C-terminal fragment of drosulfakinin II (DrmSKII), Asp-Gln-Phe-Asp-Asp-Tyr(SO₃H)-Gly-His-Met-Arg-Phe-NH₂, has been studied in the blowfly Calliphora vomitoria. Antisera were preabsorbed with combinations of the parent antigen, the tetrapeptide Phe-Met-Arg-Phe-NH₂ and cholecystokinin, the vertebrate sulfated octapeptide (CCK-8), Asp-Tyr(SO₃H)-Met-Gly-Trp-Met-Asp-Phe-NH₂, in order to ensure specificity for the sulfakinin peptides of C. vomitoria (the nonapeptide callisulfakinin I is identical to drosulfakinin I and callisulfakinin II differs from DrmSK II only by the presence of -Glu³-Glu⁴- in place of -Asp³-Asp⁴-). Only four pairs of sulfakinin-immunoreactive neurones have been visualised in the entire nervous system. These occur in the brain: two pairs of cells situated medially in the caudo-dorsal region close to the roots of the ocellar nerve and two other pairs at the same level but positioned more laterally. Despite the small number of sulfakinin-immunoreactive cells, there are extensive projections to many areas of neuropile in the brain and the thoracic ganglion. The pathway of the medial sulfakinin cells extends into each of the three thoracic ganglia and a metameric arrangement of sulfakinin neuronal projections is also seen in the abdominal ganglia. Neither the dorsal neural sheath of the thoracic ganglion, nor the abdominal nerves contain sulfakinin-immunoreactive material. These observations suggest that the sulfakinins of the blowfly function as neurotransmitters or neuromodulators. They do not appear to have a direct role in gut physiology, as has been shown by in vitro bioassays for the sulfakinins of orthopterans and blattodeans. In addition to the neurones that display specific sulfakinin immunoreactivity, other cells within the brain and thoracic ganglion are immunoreactive to cholecystokinin/gastrin antisera. There are, therefore, at least two types of dipteran neuropeptides with amino acid sequences that are similar to the vertebrate molecules cholecystokinin and gastrin.     

Mass Spectrometric Studies of the Effect of pH on the Accumulation of Intermediates in Denitrification by Paracoccus denitrificans

We have used a quadrupole mass spectrometer with a gas-permeable membrane inlet for continuous measurements of the production of N₂O and N₂ from nitrate or nitrite by cell suspensions of Paracoccus denitrificans. The use of nitrate and nitrite labeled with ¹⁵N was shown to simplify the interpretation of the results when these gases were measured. This approach was used to study the effect of pH on the production of denitrification intermediates from nitrate and nitrite under anoxic conditions. The kinetic patterns observed were quite different at acidic and alkaline pH values. At pH 5.5, first nitrate was converted to nitrite, then nitrite was converted to N₂O, and finally N₂O was converted to N₂. At pH 8.5, nitrate was converted directly to N₂, and the intermediates accumulated to only low steady-state concentrations. The sequential usage of nitrate, nitrite, and nitrous oxide observed at pH 5.5 was simulated by using a kinetic model of a branched electron transport chain in which alternative terminal reductases compete for a common reductant.