Macrozoobenthos of three Pennsylvania lakes: responses to acidification

The littoral macrozoobenthos (MZB) of three northeastern Pennsylvania lakes was sampled seasonally from summer 1981 until summer 1983, to determine if any changes were occurring in response to acid deposition. In the acidified lake (total alkalinity 0.0 eq L^–1) the mean pH decreased from 5.5 in 1981 to 4.2 in 1983. Chironomidae comprised 71.30% of the MZB numbers and 19.6% of the wet weight. Over the study period the wet weight of Chironomidae increased (p < 0.04) as did the total numbers of Chironomidae in general (p < 0.01) and Tanytarsini (p < 0.01) in particular. Total numbers of MZB also increased (p < 0.02) in the acidified lake, but there was no significant change in the number of taxa, diversity or total wet weight. In the moderately sensitive lake (total alkalinity 47.4 eq L^–1, mean pH 6.1) Chironomidae were numerically (43%) dominant but Odonata (18.6%) and Mollusca (12.7%) dominated wet weight. There were no significant changes in the MZB of the moderately sensitive lake over the study period. In the least sensitive lake (total alkalinity 190 eq L^–1, mean pH 6.6) the Amphipoda (31.3%) and Chironomidae (27.3%) together provided 58.6% of the MZB numbers, and the Mollusca formed 55.1% of wet weight. Wet weight at the least sensitive lake was higher (p < 0.01) and there were more Ephemeroptera, Pelecypoda and Gastropoda than at the other two lakes. There were no differences in total numbers, diversity or number of taxa among the three lakes.     

Selection of DNA binding sites by regulatory proteins. Statistical-mechanical theory and application to operators and promoters

We present a statistical-mechanical selection theory for the sequence analysis of a set of specific DNA regulatory sites that makes it possible to predict the relationship between individual base-pair choices in the site and specific activity (affinity). The theory is based on the assumption that specific DNA sequences have been selected to conform to some requirement for protein binding (or activity), and that all sequences that can fulfil this requirement are equally likely to occur. In most cases, the number of specific DNA sequences that are known for a certain DNA-binding protein is very small, and we discuss in detail the small-sample uncertainties that this leads to. When applied to the binding sites for cro repressor in phage lambda, the theory can predict, from the sequence statistics alone, their rank order binding affinities in reasonable agreement with measured values. However, the statistical uncertainty generated by such a small sample (only 6 sites known) limits the result to order-of-magnitude comparisons. When applied to the much larger sample of Escherichia coli promoter sequences, the theory predicts the correlation between in vitro activity (k2KB values) and homology score (closeness to the consensus sequence) observed by Mulligan et al. (1984). The analysis of base-pair frequencies in the promoter sample is consistent with the assumption that base-pairs at different positions in the sites contribute independently to the specific activity, except in a few marginal cases that are discussed. When the promoter sites are ordered according to predicted activities, they seem to conform to the Gaussian distribution that results from a requirement for maximal sequence variability within the constraint of providing a certain average activity. The theory allows us to compare the number of specific sites with a certain activity to the number that would be expected from random occurrence in the genome. While strong promoters are "overspecified", in the sense that their probability of random occurrence is very low, random sequences with weak promoter-like properties are expected to occur in very large numbers. This leads to the conclusion that functional specificity is based on other properties in addition to primary sequence recognition; some possibilities are discussed. Finally, we show that the sequence information, as defined by Schneider et al. (1986), can be used directly (at least in the case of equilibrium binding sites) to estimate the number of protein molecules that are specifically bound at random "pseudosites" in the genome.(ABSTRACT TRUNCATED AT 400 WORDS)     

Measurement of Acetylcholine Release in Freely Moving Rats by Means of Automated Intracerebral Dialysis

The present study demonstrates the feasibility of measuring acetylcholine in perfusion samples collected by means of in vivo brain dialysis in the striata of freely moving rats. The output of the dialysis device was directly connected to an automated sample valve of a HPLC-assay system that comprises a cation exchanger, a post-column enzyme reactor, and an electrochemical detector. The presence of an acetylcholinesterase inhibitor (neostigmine) in the perfusion fluid was required for the detection of acetylcholine in the perfusate. Increasing concentrations of neostigmine induced increasing amounts of acetylcholine. Continuous perfusion with a fixed concentration (2 microM) of neostigmine resulted in gradually increasing amounts of collected acetylcholine over time although a considerable variation between successive samples exists. The brain dialysis technique was further validated by studying the effect of various drugs. Systemically administered atropine increased the output of acetylcholine, whereas the addition of tetrodotoxin to the perfusion fluid resulted in a complete disappearance of the neurotransmitter.     

Determination of small quantities of sulfate (0-12 nmol) in serum, urine, and cartilage of the mouse

The colorimetric benzidine method of K. S. Dodgson and B. Spencer (1953, Biochem. J. 55, 436-440) for the measurement of inorganic sulfate can be scaled down about 100 times by using disposable 96-well microplates instead of individual cuvettes. Ten-microliter samples of serum and urine, derived from mice, can be analyzed in a simple, rapid, and reliable way without sacrificing the animals. Without prior isolation of sulfated glycosaminoglycans, ester sulfate in mouse patellar cartilage is liberated quantitatively as inorganic sulfate upon acid hydrolysis in 3 M HCl for 16 h at 80 degrees C. To this end the articular cartilage layer of the patella must be separated in toto from the underlying bone. Subsequent hydrolysis in polypropylene tubes gives accurate results. In contrast, hydrolysis in borosilicate glass vials is useless, since nanomoles of sulfate added cannot be recovered adequately. The thin patellar cartilage layer obtained from 10-week-old male mice contains about 5 nmol of sulfate, an amount easily measured with the developed microplate benzidine method.     

Intracellular transport and degradation of chylomicron remnants in rat liver cells after in vivo endocytosis

The intracellular transport and degradation of in vivo endocytosed chylomicron remnants labelled with 125I in the protein moiety was studied in rat liver cells by means of subcellular fractionation in Nycodenz and sucrose density gradients. Initially, the radioactivity was located in low-density endosomes and was sequentially transferred to light and dense lysosomes. Data from gel filtration of the light and dense lysosomal fractions showed radioactive material with a molecular weight of about 1000-2000, representing short peptide fragments or amino acids which remain attached to iodinated tyramine cellobiose. In addition, undegraded apoproteins accumulated in both types of lysosome. Our data suggest that endocytosed chylomicron remnant apoproteins are first located in low-density endosomes and are sequentially transferred to light and dense lysosomes. Furthermore, the degradation process starts in the light lysosomes.     

Further antigenic analyses of the fish pathogenic bacteriumVibrio anguillarum

TenVibrio anguillarum strains were selected for an immunoelectrophoretic study. Evidence was provided for existence of two new K antigens which displayed cross-reactivity. The importance of an exact characterization of surface antigens inV. anguillarum is considered.     

Linkage relationship between the genes for adenosine deaminase and S-adenosyl-homocysteine hydrolase on human chromosome 20

Summary The genes for adenosine deaminase (ADA) and Sadenosyl homocysteine hydrolase (AHCY or SAHH) are known to be syntenic and within measurable distance from each other, on chromosome 20 in man. In the present study an informative family is described in which the recombination fraction (θ) between the respective genes is estimated to be about 0.18. Together with the published finding of θ=0.15 (Eiberg and Mohr 1985) in informative Danish families, the recombination fraction for the pooled data is calculated to be θ=0.14 (in men), (in women) and (both sexes taken together).     

Cytoplasm-enriched fragments ofChara: structure and electrophysiology

Summary Cytoplasm-enriched fragments prepared from internodal cells ofChara corallina by centrifugation contain membrane bound vesicles ranging in size from a few m to hundreds of m. If the fragments are incubated in artificial pond water (APW) of pH₀ above 6.5, neutral red stains the inside of many vesicles bright crimson, suggesting the presence of inward proton-pumping. In APW of pH₀ below 6 crimson vesicles are found less frequently. Under such conditions most vesicles remain unstained inside and some develop indistinct pink halos. After a few days most fragments form a central vacuole, which stains red, regardless of the pH₀. The cytoplasmic layer still contains vesicles after vacuole formation.In order to identify the membrane bounding the vesicles various fluorescent probes were applied either by injection into the fragment or directly onto the vesicles released into artificial cytoplasm. Lucifer yellow or 6 COOH-F move readily across the tonoplast in intact cells, but did not enter any vesicles. On the other hand, the fluorescent cationic stain DIOC, which is used to highlight mitochondria and especially endoplasmic reticulum, stained the vesicle membrane. Numerous elliptical or kidney shaped nuclei in the flowing cytoplasm were highlighted with DAPI. In some fragments the nuclei formed large agreggates sometimes filling the width of the fragment.Patch-clamping the vesicles in artificial cytoplasm showed the presence of several kinds of channels, some displaying similar behaviour to the K⁺ channels observed in cytoplasmic droplets.Analogous to the plasmalemma of intact cells, the fragments without vacuoles displayed electrophysiological states dominated by either K⁺ conduction, H⁺ (or OH^–) conduction or the proton pump. On the other hand, excitation transients in fragments were of low amplitude or absent altogether. Detailed comparisons of data from fragments and intact cells are shown. The effect of vacuole formation on fragment electrophysiology was also explored.