Distribution of macroinvertebrates in the shallow part of ‘De Gijster’, a water storage lake in the Biesbosch (The Netherlands)

The composition and distribution of the macroinvertebrate fauna of the shallow part of De Gijster, a medium sized water storage lake in The Netherlands, has been studied during 1984. The numerous individuals collected belong mainly to the Oligochaeta, Diptera (Chironomidae larvae) and Mollusca. Amongst them are elements of both the former polder fauna and the river fauna. A quantitative analysis revealed data on the horizontal distribution of several taxa within the lake as well as data on the influence of the lifecycle of some taxa on the annual composition of the fauna.     

Effects of fructose on human fibroblast metabolism: the application of DNA measurements as a basis for interpretation

Summary A fluorometric procedure for measuring DNA was used to study growth and metabolic responses of eight cell strains of human foreskin fibroblasts. In preliminary studies this procedure gave more precise specific activity changes inN-acetyl-β-d-glucosaminidase (NAG) than did a protein activity basis, when changes in this enzyme's specific activity were investigated as a function of experimental cell manipulation. When fibroblast growth in eight cell strains was compared in 134 mM d-fructose vs. 13.4 mM glucose-supplemented minimum essential media, a significant increase in cellular DNA (50%) and protein (45%) occurred over an 11-d period. No significant differences in media pH change, lactate production, or carbohydrate uptake occurred on a DNA basis when cell metabolism was compared over the last 24 h of culture in the two media. Cells grown in fructose-containing media tended to show a reduction in NAG specific activity when compared with those grown in glucose-containing media.     

Macrophage mediation of the inhibitory effects of elevated intracellular levels of adenosine-3':5' cyclic monophosphate (cAMP) on macrophage-Trypanosoma cruzi association

Presence of theophylline and dibutyryl-cAMP—agents which cause elevation of intracellular levels of cAMP—in in vitro systems in which murine macrophages interact with virulent blood forms of Trypanosoma cruzi resulted in a marked inhibition of cell-parasite association (i.e., decreased surface binding and/or internalization). This effect was evidenced in terms of significant reductions in both the percentage of infected macrophages and the average number of trypanosomes per 100 macrophages. Pretreatment of the macrophages with these agents produced a similar inhibition whereas pretreatment of the parasites had no significant consequence on the interaction. The inhibitory effect was transient since it was no longer seen after 30 min of incubation of the treated macrophages in fresh medium. Thus, the inhibitory effect is exerted through a transient effect on the macrophage.     

Studies on the mechanism of the human natural killer cell lethal hit: a new method for rapid physical isolation of lethally hit K562 targets and inhibition of cytolysis by reduced temperature or trypsin

A new method was developed which allows for rapid (2 min) physical isolation of viable K562 target cells after being programmed to lyse (lethally hit) by purified human natural killer (NK) cells (LGL). To achieve this K562 cells which were obtained from the 34-36% interface of discontinuous Percoll gradients and purified human NK cells (LGL) which were obtained from the (43-45% Percoll) interface were employed. Using a Ca2+ pulse method and the separation of NK-K562 conjugates with EDTA and rapid centrifugation on Percoll gradients at 4 degrees C we could physically isolate the lethally hit K562 cells from the LGL allowing the study of the events leading to their subsequent lysis. Lysis of "purified" lethally hit K562 cells occurred in the absence of Ca2+ or Mg2+ and was blocked by reduced temperature (4 degrees C), or by the protease enzyme trypsin. When lethally hit targets were held at 4 degrees C (to block lysis) then rewarmed to 37 degrees C lysis ensued but with a rate slower than that of control cells not held at 4 degrees C. These data support the concept that transfer of protease-sensitive and possibly temperature-dependent structures from the NK cell to the target is a requisite step in NK cytolysis.     

Unsuccessful induction of low-zone tolerance to bovine serum albumin injected into the subarachnoid space

Introduction of T-dependent antigens into the subarachnoid space (isas) resulted in higher systemic antibody responses in mice than injections into the peritoneal cavity (ip) or other sites commonly used for immunization. Antibody production in isas immunized mice was not increased by treatment with cyclophosphamide (Cy) at doses known to abolish T-suppressor-cell activity, but such treatment increased antibody production in ip immunized mice toward the higher level which was observed in the isas immunized animals. Suppressor cell-dependent low zone tolerance (LZT) to TNP-BSA could not be induced by isas injections of deaggregated BSA (d-BSA). Conversely, mice which were unresponsive to ip injected d-BSA showed consistent systemic antibody responses when the antigen was injected isas. These observations indicate that immune responses initiated within the CNS are associated with relatively ineffective induction of systemic suppressor cell activity.     

Differential effects of positive and negative proliferative stimuli on murine cytolytic and helper T-cell clones

Studies were performed to determine whether substances could be identified which exhibited differential regulatory effects--either positive or negative--on the growth of murine alloreactive cytolytic (Tc) and helper (Th) cloned T-cell lines. The following lines of evidence suggested that Tc and Th proliferate in response to the same growth factor (GF). (1) When GF-containing fluids from cultures of phorbol myristic acetate (PMA)-activated EL4 thymoma were fractionated by a variety of biochemical techniques. Tc and Th eluted together. (2) Absorption of GF-containing supernatants with either cloned Tc or cloned Th depleted GF activity for each to a similar extent, and GF eluted from either Tc or Th to which it had adsorbed supported the proliferation of Tc and Th equally well. (3) Lectin-depleted supernatants from cultures of concanavalin A (Con A)-activated Th stimulated the proliferation of Th as well as Tc. (4) Recombinant human interleukin (IL-2) supported the growth of Tc and Th with equal efficiency. On the other hand, the following observations indicated that Tc and Th differed in their responses to inhibitors of GF-driven proliferation. (1) Con A at greater than or equal to 0.3 micrograms/ml inhibited the GF-driven proliferation of each of three Th lines but not either of two Tc lines. To the contrary, Con A enhanced GF-dependent proliferation of Tc. (2) Like Con A, allogeneic splenocytes selectively depressed GF-driven proliferation of Th but not Tc. (3) A substance generated during the acid elution of GF from cells, possibly a modified fetal calf serum component, greatly reduced the GF-driven proliferation of Tc but not Th. These results suggest that differential control of the proliferation of Tc and Th in cellular immune responses may be achieved via negative regulatory signals and raise the possibility that substances which can selectively depress the proliferation of specific T-cell subsets might be found which would be of therapeutic value.     

Effects of glucagon on the redox states of cytochromes in mitochondria in situ in perfused rat liver

The effects of glucagon on the respiratory function of mitochondria in situ were investigated in isolated perfused rat liver. Glucagon at the concentrations higher than 20 pM and cyclic AMP (75 microM) stimulated hepatic respiration, and shifted the redox state of pyridine nucleotide (NADH/NAD) in mitochondria in situ to a more reduced state as judged by organ fluorometry and beta-hydroxybutyrate/acetoacetate ratio. The organ spectrophotometric study revealed that glucagon and cyclic AMP induced the reduction of redox states of cytochromes a(a3), b and c+c1. Atractyloside (4 micrograms/ml) abolished the effects of glucagon on these parameters and gluconeogenesis from lactate. These observations suggest that glucagon increases the availability of substrates for mitochondrial respiration, and this alteration in mitochondrial function is crucial in enhancing gluconeogenesis.     

Forward mutation assay of V79 cells to 6-thioguanine resistance in a soft agar technique that eliminates effects of metabolic co-operation

A technique involving culture in soft agar was used for the assay of forward mutation of V79 cells to 6-thioguanine (6TG) resistance. The main reason for the use of soft agar was to prevent reduction in recovery of mutants depending on the cell density plated for mutation selection, which is the chief problem in the liquid method, and which results mainly from metabolic co-operation due to cell-to-cell contact.V79 cells grew well in fortified soft agar medium (DMEM + 20% FBS) showing cloning efficiencies (>80%) as high as in liquid culture. Therefore, V79/HGPRT mutagenesis could be assayed quantitatively in soft agar culture.The frequency of 6TG-resistant colonies in agar selective medium increased linearly with increase in concentration of EMS. Toxicity and mutagenic responses were greater in soft agar than in liquid culture.In cultures of untreated and EMS-treated cells, more than 95% of the 6TG-resistant colonies isolated were aminopterin-sensitive.Use of soft agar for selection prevented the reduction in the number of mutants with increase in the size of incula on plating up to 1?2 × 10⁶ cells per 9-cm dish: in liquid culture, even with a lower plating number (2 × 10⁵ cells per 9-cm dish), a notable reduction in numbers of mutants was observed. This character was re-examined in a reconstruction experiment. The results show that, when up to 2 × 10⁶ cells were plated per 9-cm dish, 6TG-resistant cells were almost completely recovered from the soft agar medium, whereas only 10% were recovered from liquid culture.     

125I] 3-quinuclidinyl 4-iodobenzilate: a high affinity, high specific activity radioligand for the M1 and M2-acetylcholine receptors

We have prepared a radioiodinated ligand which binds with high affinity to the muscarinic acetylcholine receptor (m-AChR). A derivative of 3-quinuclidinyl benzilate, [125I] labeled (R) 1-aza-bicyclo(2.2.2)oct-3-yl (R,S)-alpha-hydroxy-alpha-(4-[125I]iodophenyl)phenyl acetate (4- IQNB ) exhibits an affinity for the m-AChR from corpus striatum higher than that of (R) [3H] QNB. Additionally, [125I] 4- IQNB exhibits receptor selectivity for the M1 receptor since the affinity for the receptor from dog and rat heart is lower than that using dog or rat corpus striatum.     

Interstrand duplexes in Friend erythroleukemia nuclear RNA. The interaction of non-polyadenylated nuclear RNA with polyadenylated nuclear RNA and with small nuclear RNAs

Intermolecular duplexes among large nuclear RNAs, and between small nuclear RNA and heterogeneous nuclear RNA, were studied after isolation by a procedure that yielded protein-free RNA without the use of phenol or high salt. The bulk of the pulse-labeled RNA had a sedimentation coefficient greater than 45 S. After heating in 50% (v/v) formamide, it sedimented between the 18 S and 28 S regions of the sucrose gradient. Proof of the existence of interstrand duplexes prior to deproteinization was obtained by the introduction of interstrand cross-links using 4'-aminomethyl-4,5',8-trimethylpsoralen and u.v. irradiation. Thermal denaturation did not reduce the sedimentation coefficient of pulse-labeled RNA obtained from nuclei treated with this reagent and u.v. irradiated. Interstrand duplexes were observed among the non-polyadenylated RNA species as well as between polyadenylated and non-polyadenylated RNAs. beta-Globin mRNA but not beta-globin pre-mRNA also contained interstrand duplex regions. In this study, we were able to identify two distinct classes of polyadenylated nuclear RNA, which were differentiated with respect to whether or not they were associated with other RNA molecules. The first class was composed of poly(A)+ molecules that were free of interactions with other RNAs. beta-Globin pre-mRNA belongs to this class. The second class included poly(A)+ molecules that contained interstrand duplexes. beta-Globin mRNA is involved in this kind of interaction. In addition, hybrids between small nuclear RNAs and heterogeneous nuclear RNA were isolated. These hybrids were formed with all the U-rich species, 4.5 S, 4.5 SI and a novel species designated W. Approximately equal numbers of hybrids were formed by species U1a, U1b, U2, U6 and W; however, species U4 and U5 were significantly under-represented. Most of these hybrids were found to be associated stably with non-polyadenylated RNA. These observations demonstrated for the first time that small nuclear RNA-heterogeneous nuclear RNA hybrids can be isolated without crosslinking, and that proteins are not necessary to stabilize the complexes. However, not all molecules of a given small nuclear RNA species are involved in the formation of these hybrids. The distribution of a given small nuclear RNA species between the free and bound state does not reflect the stability of the complex in vitro but rather the abundance of complementary sequences in the heterogeneous nuclear RNA.(ABSTRACT TRUNCATED AT 400 WORDS)