Aquatic ecology in The Netherlands: What is being done by whom?

Summary About one third of the Dutch environmental research is concentrated on aquatic problems. The largest number of these projects is physicochemical (65%) and 47% of the aquatic projects contains an ecological component. The aquatic research projects are executed in a large number of different research institutes. Many institutes just formulated one project (43.2%) and about 10% of the institutes formulated 10 projects. The institutes with 10 or more projects account for almost 50% of the total number of projects. However, the size of the research projects with respect to the average total personnel per year may differ considerably. The largest number of aquatic research projects is carried out in governmental institutes. This relative high share of governmental institutes has proportionally increased during the period of 1975–1982. During this period the relative shares of all the aquatic research projects with an ecological component, of the strictly ecological projects and of the ecological/physicochemical projects have also proportionally inclined. However, in absolute numbers there seems to be a decline of both ecological and non-ecological projects on aquatic problems.     

Spontaneous insertion of plant plasma membrane (H+)ATPase into a preformed bilayer

Summary The purified (H⁺ATPase from corn roots plasma membrane inserted spontaneously into preformed bilayer from soybean lipids. The yield of the protein insertion, as measured from its H⁺-pumping activity, increased as a function of lipids and protein concentrations. In optimum conditions, all the (H⁺)ATPase molecules were closely associated with liposomes, exhibiting a high H⁺-pumping activity (150,000% quenching· min^–1·mg^–1 protein of the probe 9-amino-6-chloro-2-methoxyacridine). The insertion was achieved within a few seconds. No latency of the (H⁺)ATPase hydrolytic activity was revealed when lysophosphatidylcholine was added to permeabilize the vesicles. This indicated that the (H⁺)ATPase molecules inserted unidirectionally, the catalytic sites being exposed outside the vesicles (inside-out orientation), and thus freely accessible to Mg-ATP. The nondelipidated (H⁺)ATPase could also functionally insert into bilayer from PCPEPG or PCPEPI, due to the presence of both hydrophobic defects promoted by PE, and negative phospholipids specifically required by the (H⁺)ATPase from corn roots. The detergent octylglucoside facilitated the delipidated (H⁺)ATPase reinsertion probably by promoting both a proper protein conformation and hydrophobic defects in the bilayer. Lysophosphatidylcholine facilitated the delipidated protein insertion only when hydrophobic defects were already present, and thus seemed only capable to ensure a proper protein conformation     

cDNA and amino acid sequences of rainbow trout (Oncorhynchus mykiss) lysozymes and their implications for the evolution of lysozyme and lactalbumin

Summary The complete 129-amino-acid sequences of two rainbow trout lysozymes (I and II) isolated from kidney were established using protein chemistry microtechniques. The two sequences differ only at position 86, I having aspartic acid and II having alanine. A cDNA clone coding for rainbow trout lysozyme was isolated from a cDNA library made from liver mRNA. Sequencing of the cloned cDNA insert, which was 1 kb in length, revealed a 432-bp open reading frame encoding an amino-terminal peptide of 15 amino acids and a mature enzyme of 129 amino acids identical in sequence to II. Forms I and II from kidney and liver were also analyzed using enzymatic amplification via PCR and direct sequencing; both organs contain mRNA encoding the two lysozymes. Evolutionary trees relating DNA sequences coding for lysozymesc and α-lactalbumins provide evidence that the gene duplication giving rise to conventional vertebrate lysozymesc and to lactalbumin preceded the divergence of fishes and tetrapods about 400 Myr ago. Evolutionary analysis also suggests that amino acid replacements may have accumulated more slowly on the lineage leading to fish lysozyme than on those leading to mammal and bird lysozymes.     

Morphology and dynamics of protruding spirochete periplasmic flagella.

We recently characterized the three-dimensional shape of Treponema phagedenis periplasmic flagella (PFs). In the course of these studies, we observed protrusions on swimming cells that resembled PFs. Here we present a detailed characterization of the shape, structure, and motion of these protrusions. Although protrusion formation occurred primarily in wild-type cells during the stationary phase, a large fraction of exponential-phase cells of cell cylinder helicity mutants (greater than 90% of mutant T-52) had protrusions. These results suggest that cells bearing protrusions can still participate in cell division. T. phagedenis protrusions had the identical helix handedness, pitch, and diameter to those of purified PFs. Protrusions were not present on mutants unable to synthesize PFs, but were present in all motile revertants which regained PFs. These results, taken together with electron microscope observations, suggest that protrusions consist of PFs surrounded by an outer membrane sheath. To analyze protrusion movements, we held cells against a coverglass surface with optical tweezers and observed the motion of protrusions by video-enhanced differential interference contrast light microscopy. Protrusions were found to gyrate in both clockwise and counterclockwise directions, and direct evidence was obtained that protrusions rotate. Protrusions were also observed on Treponema denticola and Borrelia burgdorferi. These were also left-handed and had the same helix handedness, pitch, and diameter as purified PFs from their respective species. The PFs from T. denticola had a helix diameter of 0.26 microns and a helix pitch of 0.78 micron; PFs from B. burgdorferi had a helix diameter of 0.28 micron and a helix pitch of 1.48 microns. Protrusions from these spirochete species had similar structures and motion to those of T. phagedenis. Our results present direct evidence that PFs rotate and support previously proposed models of spirochete motility.     

Amino acids are general growth inhibitors of Nicotiana silvestris in tissue culture

The growth of Nicotiana silvestris in suspension culture is inhibited by all of the common protein amino acids at the millimolar level, except for L-glutamine. A defined experimental system for growth/inhibition studies has been established, and growth studies were carried out with cells that had been maintained in the exponential growth phase for at least 10 generations (EE cells). The following results were obtained after particularly detailed studies with aromatic amino acids. The onset of inhibition was preceded by a duration of normal growth rate which varied within a range of 12 to 48 h. The degree of inhibition was directly proportional to amino acid concentration and inversely related to the initial cell density of the inoculum. A slowed, but still exponential rate of growth persisted during an early phase of inhibition. Under sufficiently severe conditions, this was followed by progressive diminution of growth rate and eventual lysis. The most drastic inhibitory effects caused by aromatic amino acids were in the order: phenylalanine, tryptophan and tyrosine. When EE cells cultivated under conditions of growth inhibition were diluted into fresh medium, immediate resumption of growth at the uninhibited rate occurred and persisted. On the other hand, when growth-inhibited EE cells were diluted into medium containing the same concentration of amino acid used in the first round of growth, an initial burst of uninhibited growth lasting about 24 h was followed by a drastic, progressively declining growth rate which deteriorated to cell death and lysis. When cells in stationary phase were used as an inoculum, as is done in typical growth characterizations with suspension cultures, the sensitivity to inhibition during the subsequent exponential growth phase was several-fold greater than was the case with EE cells. Hypotheses that growth inhibition might be caused by ammonia toxicity, keto-acid toxicity, or by inhibition of nitrate utilization were ruled out. Observations that provide new insight are: (i)growth-inhibited cells undergo drastic plasmolysis, (ii) L-glutamine is an effective antagonist of amino-acid inhibitors, and (iii) growth-inhibited cells exhibit a transient restoration of normal growth rate upon dilution into fresh growth medium. These results implicate a linkage of amino acids with osmotic regulation and nitrogen metabolism.     

The silk-producing system ofLinyphia triangularis (Araneae,Linyphiidae) and some comparisons with Araneidae

Summary The spinning apparatus ofLinyphia triangularis, adult females and males, was studied with the scanning electron microscope and the main anatomical and histochemical characteristics of the silk glands, including the epigastric apparatus of males, are presented. The epigastric glands seem to be important for the construction of sperm webs. A detailed account of the use of the different kinds of silk in web building is given.The spinning apparatus ofLinyphia closely corresponds to the araneid pattern. Characteristic of linyphiid spiders is the poor development of the aciniform glands. Corresponding to the minor importance of capture threads forLinyphia, the triads (aggregate and flagelliform glands) are less developed than in Araneidae.Linyphia make much less use of the secretions of the piriform glands for connecting threads than Araneidae. Capture threads adhere to other threads by their own glue; other threads seem mostly to be bound to one another by the secretion of the minor ampullate glands whose chemical properties, inLinyphia, appear especially adapted to this function. Neither the anatomical and histochemical data concerning the spinning apparatus nor the structure of the webs provide any indication of close relationships between Linyphiidae and Agelenidae, as was recently claimed.     

Effects of diphenyl ether herbicides on porphyrin accumulation by cultured hepatocytes

Several diphenyl ether herbicides, such as acifluorfen methyl, have been previously shown to cause large accumulations of the heme and chlorophyll precursor, protoporphyrin, in plants. Lightinduced herbicidal damage is mediated by the photoactive porphyrin. Here we investigate whether diphenyl ether herbicides can affect porphyrin synthesis in rat and chick hepatocytes. In rat hepatocyte cultures, protoporphyrin, as well as coproporphyrin, accumulated after treatment with acifluorfen or acifluorfen methyl. Combination of acifluorfen methyl with an esterase inhibitor to prevent the conversion of acifluorfen methyl to acifluorfen resulted in a greater accumulation of porphyrins than caused by acifluorfen methyl or acifluorfen alone. In vitro enzyme studies of hepatic mitochondria isolated from rat and chick embryos demonstrated that protopor-phyrinogen oxidase, the penultimate enzyme of heme biosynthesis, was inhibited by low concentrations of acifluorfen, nitrofen, or acifluorfen methyl with the latter being the most potent inhibitor. These findings indicate that diphenyl ether treatment can cause protoporphyrin accumulation in rat hepatocyte cultures and suggest that this accumulation was associated with the inhibition of protoporphyrinogen oxidase. In cultured chick embryo hepatocytes, treatment with acifluorfen methyl plus an esterase inhibitor caused massive accumulation of uroporphyrin rather than protoporphyrin or coproporphyrin. Specific isozymes of cytochrome P450 were also induced in chick embryo hepatocytes. These effects were not observed in the absence of an esterase inhibitor. These results suggest that diphenyl ether herbicides can cause uroporphyrin accumulation similar to that induced by other cytochrome P450-inducing chemicals such as polyhalogenated aromatic hydrocarbons in the chick hepatocyte system.     

Calcium-sensitive cells of the pars intermedia and osmotic balance in the eel

Summary The structure of the PAS-positive calcium-sensitive (Ca-s) cells of the pars intermedia was investigated in eels kept in deionized water (DW) or fresh water (FW) supplemented with Ca²⁺ or Mg²⁺. Ca²⁺ (2mM) reduces considerably the response to DW; plasma osmolarity, Na⁺ and Ca²⁺ levels are not significantly affected. In eels adapted to DW for 21 or 28 days, showing highly stimulated Ca-s cells, an addition of CaCl₂ for 2 days inhibits the release of granules, but does not immediately block their synthesis and the mitotic activity. The nuclear area is reduced, osmolarity and plasma sodium increase, but the rise in calcium is not always significant. Magnesium, at a 10-fold greater concentration than in FW (2 mM), slightly inhibits the release of secretory granules without reducing other indicators of stimulation. In Ca-enriched FW, the Ca-s cells appear inactive. These data show that the PAS-positive cells in the pars intermedia of the eel are calcium-sensitive, similar to those of the goldfish; their role in calcium regulation is briefly discussed.     

Distribution of lipid synthesizing enzymes, 2′,3′-cyclic nucleotide 3′-phosphodiesterase,and myelin proteins in rat forebrain subfractions during development

The distribution of UDP-galactose: ceramide galactosyltransferase (CGalT) was studied in subcellular fractions of rat forebrain during development using zonal centrifugation on linear gradients. Specialized subfractions: SN 1, a microsomal fraction, SN 4, a myelin-related fraction, and purified myelin were also used for this study. For comparison, two microsomal lipid synthesizing enzymes, a myelin-specific enzyme, 2,3-cyclic nucleotide 3-phosphodiesterase and myelin proteins were measured in the same subfractions. UDP-glucose: ceramide glucosyltransferase and cerebroside sulfotransferase were confined to microsomes. CGalT was ferase and cerebroside sulfotransferase were confined to microsomes. CGalT was localized in microsomes, but also in myelin and myelin-related fractions. The developmental change in distribution of CGalT in adult animals toward myelin containing fractions could indicate that the replacement of galactosylceramide in compact myelin could be carried out in close proximity to compact myelin (mesaxon, paranodal loops) rather than in the distant oligodendrocyte perikaryon.     

Characterization ofκ-casein and keratin domains in fibrinogen

Summary Following the observation of a close sequence homology between the N-terminal moiety of the -chain of fibrinogen with large parts of-casein, the occurrence of a keratin domain in the middle section of the Achain is suggested.