Effects of pyrophosphate, triphosphate, and potassium chloride on adenylate deaminase from rat muscle

Inorganic pyrophosphate and triphosphate inhibit adenylate deaminase from rat skeletal muscle with K1 values of 10 and 1.5 microM, respectively, in the presence of 150 mM KCl at pH 7. They act by reducing the apparent affinity of the enzyme for AMP, with relatively small effects on Vmax. The inhibitions are diminished by H+, the KI values increasing two- to threefold in going from pH 7.0 to 6.2, and are relieved by ADP. These properties are similar to the inhibitions produced by GTP and ATP, indicating that pyrophosphate and triphosphate act like analogues of the nucleoside triphosphates. Neither of these inhibitors shows relief of inhibition at high concentrations as do ATP and GTP. These results suggest that nucleotides interact with the inhibitor site of the enzyme primarily through their phosphate moieties and with the activator site primarily through their nucleoside moieties. As the concentration of KCl is increased from 25 to 300 mM, the apparent affinities of the enzyme for ATP, GTP, orthophosphate, pyrophosphate, and triphosphate are decreased 8-100-fold. The cooperativity of the inhibitions is increased with the Hill coefficient rising from 1.0 to 1.3-1.8, and the maximum inhibition approaches 100%. Maximum activation by ADP is reduced from 1800% at 25 mM KCl to 80% at 200 mM KCl. Experiments with (CH3)4NCl indicate that activation of the enzyme by KCl involves both specific K+ effects and ionic strength effects.     

Hybrids of bleak, Alburnus alburnus, and chub, Leuciscus cephalus in English rivers

The occurrence of hybrids between bleak, Alburnus alburnus , and chub, Leuciscus cephalus is reported from four discrete river systems in England. The hybrids are described and illustrated, and means of identification from the parent species are given. Bleak × chub hybrids appear to be moderately common and on superficial examination resemble the bleak parent species more than the chub.     

Ictalurus melas (Rafinesque, 1820) and I. nebulosus (Lesueur, 1819): the North American catfishes in Europe

The greater part of the literature on European fishes reports the widespread occurrence of an introduced North American catfish and identifies it as Ictalurus nebulosus. Study of the literature reporting critical determinations and of specimens from Europe and Great Britain reveals the presence of two species, I nebulosus and I melas. These fishes are widely used in experimental studies, usually being obtained through aquarium-fish dealers indirectly from continental Europe. Mostly they are incorrectly identified as I nebulosus. There is reason to believe that I melas is the more commonly imported catfish in Britain; both species occur feral in mainland Europe.     

The assessment of cellular proliferation by immunohistochemistry: A review of currently available methods and their applications

Summary Immunohistochemical methods using antibodies to cell cycle-related antigens may be used as a means of assessing various aspects of proliferation in tissue, and have the important advantage of preserving the spatial orientation of proliferating cells in histological sections. Currently, the most widely available antibodies for this purpose are antibodies to bromodeoxyuridine (BrdU), Ki67 and antibodies to proliferating cell nuclear antigen (PCNA). BrdU is a thymidine analogue incorporated during the S phase of the cell cycle, which can be introduced by in vivo administration or by in vitro incubation, and monoclonal antibodies are available to display its localization. Ki67 demonstrates a nuclear antigen expressed in all phases of the cell cycle, except G₀ and early G₁, but can only be applied to frozen tissue. PCNA is a nuclear antigen which is essential for DNA synthesis, two commercially available antibodies to PCNA work in paraffin-embedded tissue, but may have different staining characteristics under different conditions of fixation. The main advantages and disadvantages of these different techniques are discussed, together with their main applications to date.     

Gene expression during 3T3-L1 adipocyte differentiation. Characterization of initial responses to the inducing agents and changes during commitment to differentiation.

The mouse 3T3-L1 fibroblastic cell line rapidly differentiates to an adipocyte phenotype when post-confluent cells are treated for 48 h in fetal calf serum-containing medium supplemented with 1 microM dexamethasone (D), 0.5 mM methylisobutylxanthine (M) and 10 micrograms/ml insulin (I). D and I act synergistically to commit the cells to differentiate 24-48 h after initiating treatment, and this is blocked by the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate. In order to identify cellular proteins involved in the differentiation process we analyzed differentiating 3T3-L1 cells using two-dimensional electrophoresis on large format gels. We observed changes in over 300 proteins during differentiation (over 100 within 5 h of initiating differentiation) and many of these are also changed at the level of mRNA (by analysis of in vitro translation products). About 75% of the initial changes were maximally induced by treatment with a combination of M and I, while no more than 10 proteins and their corresponding mRNAs were maximally induced by D within 3.5 h. Another 10 proteins were synergistically regulated by the combination of all three agents (DMI) within 3.5 h. Additional species were induced at later times. Five of these were synergistically induced by treatments that lead to differentiation, were first expressed at elevated levels during commitment and remained elevated in fully differentiated adipocytes. One or more of these proteins could well have a functional role in the commitment to and/or expression of the adipocyte differentiation program.     

Effect of aluminium on the growth of 34 plant species: A summary of results obtained in low ionic strength solution culture

The results from many experiments conducted over 5 years to determine the tolerance of 34 plant species (87 cultivars) to aluminium (Al) are summarised. All experiments were conducted in a temperature-controlled glasshouse using a low-ionic-strength solution culture technique. The activity of Al³⁺ (M) at which top yields were reduced by 50% (Al_RY50) was determined for each cultivar.The species Bromus wildenowii, Cynosurus cristatus, Hordeum vulgare, Triticum aestivum (cvs Warigal, Scout, Sonora-63), Avena byzantina, Arabidopsis thaliana, Lycopersicon esculentum and Nicotiana plumbaginifolia were all very sensitive to Al (Al_RY50<1). The species Poa pratense, Lolium perenne (NZ-derived cultivars), Lotus corniculatus, Avena sativa (cvs West, Carbeen, Camellia and Coolabah), Triticum aestivum (cvs Cardinal and Waalt), Allium cepa and Asparagus officinalis were sensitive to Al (Al_RY50 1–2).The pasture grass species Lolium perenne (Australian and European and derived cultivars), Lolium hybridum and Lolium multiflorum, Dactylis glomerata (Apanui and Kara), Phalaris aquatica, Festuca arundinacea and the pasture legumes species Trifolium pratense, Trifolium repens and Trifolium subterraneum were all moderately sensitive to Al (Al_RY50 2–5). Other species that were also moderately sensitive included Triticum aestivum (cvs Atlas-66, BH146, and Carazinho), Avena sativa (cvs Swan and Blackbutt), Avena Strigosa, Petunia x and Phaseolus vulgaris (cvs Red Kidney, Black Turtle and Haricot).The most tolerant species (Al_RY50>5) were (in order of increasing tolerance) Phaseolus vulgaris (cvs Tendergreen, The Prince and Yatescrop), Cucurbita maxima, Dactylis glomerata (cv Wana), Paspalum dilatatum, Lotus pedunculatus, Ehrharta calycina, Medicago sativa, Holcus lanatus, Festuca rubra, Phaseolus lunatus and Agrostis tenuis. Agrostis tenuis was at least twice as tolerant as the next most tolerant species (Al_RY50>30 compared to 15.6).     

Identification of vitellogenin in the ant, Camponotus festinatus: changes in hemolymph proteins and fat body development in workers.

Vitellogenin has been identified in the ant Camponotus festinatus, both in queens and workers. In the workers, it is already present before adult eclosion in low concentrations (less than 1 microgram/microliter hemolymph). Vitellogenin and vitellin are immunologically identical and are composed of a single type of apoprotein with an apparent Mr = 185,000. The molecular weight of the native molecules was estimated as approximately 460,000 by pore limiting gradient electrophoresis. Vitellogenin was detected as a major protein in the hemolymph of young workers, both under queenright and queenless conditions. Thus, in spite of their sterility in the presence of the queen, C. festinatus workers are able to synthetize vitellogenin which is identical both in size and immunologically to the queen vitellogenin. About 6-7 weeks after adult eclosion, however, vitellogenin was usually undetectable in the hemolymph of queenright workers, particularly the minor workers, while it constituted about 30% of total protein in queenless workers. Protein concentration in the hemolymph of queenless insects increased up to 20-fold as compared to 1-day-old insects. Queenless workers also developed large amounts of perivisceral fat body, while queenright workers, particularly the minor workers, showed a dramatic fat body regression about 6 weeks after emergence.     

Chromosomal assignment of the human genes coding for the major proteins of the desmosome junction, desmoglein DGI (DSG), desmocollins DGII/III (DSC), desmoplakins DPI/II (DSP), and plakoglobin DPIII (JUP)

We have established PCR assays for the genes coding for the major proteins of the desmosome type of cell junction, the desmosomal cadherins DGI (desmoglein) and DGII/III (desmocollins), and the plaque proteins DPI/II (desmoplakin) and DPIII (plakoglobin) and used them to test human-mouse and human-rat somatic cell hybrids with different contents of human chromosomes. From these data we were able to assign DGI to chromosome 18 (DSG), DGII/III to chromosome 9p (DSC), DPI/II to chromosome 6p21-ter(DSP), and DPIII to chromosome 7 (JUP).     

Toxicity of sewage-contaminated sediment cores to Macrocystis pyrifera (Laminariales,Phaeophyta) gametophytes determined by digital image analysis

Macrocystis pyrifera gametophytes were exposed in batch culture to varying mass concentrations of buried, sewage-contaminated, historically discharged sediment that had been sampled from two sites off Palos Verdes Peninsula, California. Significant gametophytic vegetative growth inhibition was detected in six days, using digital image analysis at sediment loadings ranging from 0.15 to 14.5 g in 500 mL nutrient-enriched seawater. Inhibition declined at low sediment loadings and increased at high loadings as cultures aged. Sediments corresponding to the historic emissions peak taken 2 km from the Joint Water Pollution Control Plant outfall inhibited vegetative growth more than did sediments sampled 13 km distant. Analysis showed elevated aqueous Cd(II), Cr(II) and p,p-DDE concentrations in high sediment-loading culture medium. Inhibition by Zn(II) alone was observed at similar concentrations in other experiments, but synergism or antagonism by other toxicants remains possible.     

Uncompetitive inhibition of monoterpene cyclases by an analog of the substrate geranyl pyrophosphate and inhibition of monoterpene biosynthesis in vivo by an analog of geraniol

Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of the acyclic precursor geranyl pyrophosphate to a variety of monocyclic and bicyclic monoterpene skeletons. Examination of the kinetics of inhibition of cyclization by the pyrophosphate ester of (E)-4-[2-diazo-3-trifluoropropionyloxy]-3-methyl-2-buten-1-o l, a photolabile structural analog of the substrate, using a partially purified preparation of geranyl pyrophosphate:(+)-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from common sage (Salvia officinalis) evidenced (under dark conditions) strictly uncompetitive inhibition with K'i values of 3.2 and 4.7 microM, respectively. These values are close to the corresponding Km values for the substrate with these two enzymes. This novel property of the substrate analog was also examined in the presence of two other inhibitors which bind to different domains of the cyclase active site (inorganic pyrophosphate and a sulfonium ion analog of a cyclic carbocationic intermediate of the reaction sequence (dimethyl-(4-methylcyclohex-3-en-1-yl)sulfonium iodide)) in order to address the mechanistic origins of the uncompetitive inhibition of cyclization. It was not possible, however, to rule out either an induced-fit mechanism or a sequential binding mechanism since the substrate is recognized by at least two binding domains and because direct examination of the effects of binding on cyclase conformation is currently not feasible. The substrate analog, although photoactive, did not give rise to light-dependent enzyme inactivation of greater magnitude than that obtained from ultraviolet light alone. The unusual behavior of the analog was attributed to intramolecular interaction of the electron-rich carbonyl group of the diazoester with the required divalent metal ion that is chelated by the pyrophosphate group. A photostable analog of geraniol that resembled the photoactive substrate analog in bearing a carbonyl function at C6 (6-oxo-3,7-dimethyloct-2(trans)en-1-ol) was prepared. Following foliar application to rapidly growing sage plants, this analog was seemingly activated to the corresponding pyrophosphate ester in vivo and selectively inhibited the activity of several cyclases in this tissue as evidenced by diminished production of the corresponding monoterpene end products.