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.     

Plasma uric acid levels in ethanol-fed turkey poults treated with allopurinol

Plasma uric acid levels were determined in ethanol-fed poults following administration of allopurinol. In young poults, allopurinol at a dose of 50 mg/kg significantly depressed plasma uric acid levels 6 hr post-dosing. At 11 hr post-dosing, plasma uric acid levels were significantly elevated in the allopurinol-treated poults when compared with control poults. During a period of ethanol abstinence, allopurinol at a dose of 40 mg/kg significantly depressed plasma uric acid levels up to 8 hr post-dosing. At a dose of 30 mg/kg, plasma uric acid levels were similar to control values at 4 and 6 hr post-dosing. Data suggest that plasma uric acid levels can be depressed in ethanol poults when allopurinol is administered every 8 hr at a dose of 40-50 mg/kg of body weight.     

Measurement of the transit time for cells through the epidermis and stratum corneum of the mouse and guinea-pig

Abstract A new approach to determine the transit time through the epidermis is presented, involving a gentle washing of the skin surface to collect the loosely attached surface corneocytes. This, it is believed, will be less likely to stimulate the system than tape-stripping or scraping. Radioactively labelled thymidine and iododeoxyuridine have been used to label cells in the basal layer and various labelled amino acids (glycine, cystine and methionine) have been used to label the metabolically viable cell layers (up to and including the granular layer). The resulting changes in surface radioactivity levels have been interpreted to provide a basal to surface transit time of 8–9.5 days for hairless and haired mouse epidermis and about 13.5 days for guinea-pigs. The basal to granular layer transit time, which probably includes some basal layer residence time, is about 4.5 days in the mouse and 8 days in the guinea-pig. The granular to surface time in mice is about 5 days. The results also suggest that when nuclear and cytoplasmic organelles are degraded in the granular layer, material is released that can diffuse rapidly through the stratum corneum to the surface. Some of this can be shown by chromatography to be thymidine. Hence, the stratum corneum is pervious to molecules such as nucleosides. This rapid diffusion outwards through the skin can also be detected shortly after injecting [¹²⁵I]-iododeoxyuridine.     

A highly sensitive chromogenic microtiter plate assay for plasminogen activators which quantitatively discriminates between the urokinase and tissue-type activators

A simple and highly sensitive chromogenic microtiter plate assay for plasminogen activators is described. The assay is based on plasmin cleavage of the synthetic tripeptide plasmin substrate H-D-norleucyl-hexahydrotyrosyl-lysine p-nitroaniline, which yields the yellow chromophore p-nitroanilide. Production of the latter compound is then quantitated spectrophotometrically at 405 nm on an ELISA plate reader. Linearity of the assay can be achieved over at least four orders of magnitude in a single experiment (0.01-100 milliPloug units) with appropriate incubation times. Capitalizing on tissue-type plasminogen activator's dependence on fibrin for enzymatic activity, the selective use of soluble fibrin products allows discrimination between urokinase and tissue-type activator. The utility of this aspect of the assay for the analysis of complex samples containing both types of plasminogen activators is demonstrated.     

An antibody to the receptor for insulin-like growth factor I inhibits the growth of MCF-7 cells in tissue culture

Alpha IR-3, a monoclonal antibody to the insulin-like growth factor I receptor which blocks insulin-like growth factor I binding and inhibits its activity, inhibits the binding of 125I-insulin-like growth factor I to MCF-7 cells (an estrogen dependent human breast carcinoma cell line) with an IC-50 of approximately 100 ng/ml. It also inhibits the growth of MCF-7 cells cultured in 5% calf serum with approximately the same IC-50. Inhibition of growth occurs both when cells are cultured in the presence and absence of estrogen and is more pronounced when cells are grown at a low density. These findings demonstrate a requirement for insulin-like growth factor I for optimal growth of MCF-7 cells and suggest that it is an autocrine growth factor in these cells.     

Labeling of bovine heart cytochrome c oxidase with analogues of phospholipids. Synthesis and reactivity of a new cardiolipin benzaldehyde probe

The syntheses of two new radioactive probes derived from cardiolipin and phosphatidylcholine are reported. These probes are derivatives of natural lipids and contain an amine-specific benzaldehyde in the head-group region. This functional group allows a choice of timing of the reaction (e.g., after equilibration and detergent removal) because an irreversible covalent bond is formed only upon the addition of reducing agent. These probes, as well as a benzaldehyde analogue of phosphatidic acid, and a water-soluble benzaldehyde reagent were covalently attached to bovine heart cytochrome c oxidase. After reconstitution into vesicles, the lipid-benzaldehyde probes selectively incorporated into the smaller polypeptides of the enzyme, while the remaining subunits (I-IV) exhibited little incorporation of label. The accessibility of amine groups labeled under the conditions used here was independent of the structural and charge differences between the benzaldehyde probes. This suggests that all three lipid probes react with polypeptides of the cytochrome c oxidase complex at general contact sites for membrane phospholipids. A water-soluble benzaldehyde reagent predominantly labeled subunits IV, Va, and Vb and polypeptides of VII-VIII. A comparison of these results facilitates a more refined view of the disposition of polypeptides of cytochrome c oxidase in respect to the lipid and aqueous phases.     

Secondary structure of 5S RNA: NMR experiments on RNA molecules partially labeled with nitrogen-15

A method has been found for reassembling fragment 1 of Escherichia coli 5S RNA from mixtures containing strand III (bases 69-87) and the complex consisting of strand II (bases 89-120) and strand IV (bases 1-11). The reassembled molecule is identical with unreconstituted fragment 1. With this technique, fragment 1 molecules have been constructed 15N-labeled either in strand III or in the strand II-strand IV complex. Spectroscopic data obtained with these partially labeled molecules show that the terminal helix of 5S RNA includes the GU and GC base pairs at positions 9 and 10 which the standard model for 5S secondary structure predicts [see Delihas, N., Anderson, J., & Singhal, R. P. (1984) Prog. Nucleic Acid Res. Mol. Biol. 31, 161-190] but that these base pairs are unstable both in the fragment and in native 5S RNA. The data also assign three resonances to the helix V region of the molecule (bases 70-77 and 99-106). None of these resonances has a "normal" chemical shift even though two of them correspond to AU or GU base pairs in the standard model. The implications of these findings for our understanding of the structure of 5S RNA and its complex with ribosomal protein L25 are discussed.     

Laser-Raman and infrared spectroscopic studies of protein conformation in the eggshell of the fish Salmo gairdneri

Laser-Raman and infrared spectroscopic studies reveal abundant beta-pleated sheet conformation in the eggshell proteins of the fish Salmo gairdneri. This secondary structure is the underlying molecular conformation, dictating the formation of the helicoidal architecture of the eggshell. Disulphide bonds crosslink the eggshell proteins of the fertilized eggs and are apparently found in g-g-g (gauche-gauche-gauche), g-g-t (gauche-gauche-trans) and t-g-t (trans-gauche-trans) conformation. There is no evidence for the existence of free sulphydryls. The tyrosines appear to act as hydrogen-bond acceptors, whereas the aromatic residues phenylalanine and tryptophan are also eggshell protein constituents.     

Endocrine responses in relation to compensatory testicular growth after neonatal hemicastration in boars

Mass (TM) and relative mass (organ mass/body mass; RTM) of the right testis and epididymis (EM and REM, respectively) were determined every 14 days from 10 to 122 days of age for intact boars (I) and boars hemicastrated on Day 10 (HC) in two crossbred herds (Trial 1 and Trial 2). Plasma follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, growth hormone (GH), and testosterone were determined in four blood samples from each pig, three collected 24 h prior to castration and one immediately prior to castration. Values for TM and RTM of HC boars were approximately double (p less than 0.0001) those of I boars by 38 days of age, and these differences were maintained through Day 122. Both EM and REM were greater (p less than 0.05) in HC than in I boars from Day 52 to Day 122. The TM, RTM, EM and REM were greater (p less than 0.05) in Trial 1 than in Trial 2 for both I and HC boars from Day 80 to Day 122, indicating an earlier onset of pubertal testicular growth in the Trial-1 boars. Plasma GH concentration was greater (p less than 0.05) in HC than in I boars from Day 16 to Day 38. A transient increase in plasma FSH (p less than 0.05) was observed from Day 24 to Day 38. After Day 38, there was no difference (p greater than 0.05) in FSH or GH between HC and I boars, or between trials. Plasma LH, prolactin, and testosterone concentrations were also similar in HC and I boars.(ABSTRACT TRUNCATED AT 250 WORDS)