Changes in choline acetyltransferase activity and high-affinity choline uptake, but not in acetylcholinesterase activity and muscarinic cholinergic receptors, in rat somatosensory cortex after sciatic nerve injury

Selected cholinergic markers (choline acetyltransferase, acetylcholinesterase, muscarinic acetylcholine receptor, high-affinity choline uptake) were studied in the hindlimb representation areas of the rat somatosensory cortex and within the visual cortex 1 to 63 days after unilateral transection of the sciatic nerve. In the contralateral somatosensory cortex, peripheral deafferentation resulted in a significant reduction of choline acetyltransferase activity (by 15%) 3 days after sciatic nerve injury, and in a significant reduction of high-affinity choline uptake (by 30%) 1 day after nerve transection, in comparison to untreated control rats. Investigations in individual cortical layers revealed that the decrease of both choline acetyltransferase activity and high-affinity choline uptake sites was mainly due to reductions in cortical layer V. Acetylcholinesterase activity and [3H]quinuclidinyl benzilate binding to muscarinic acetylcholine receptors were not affected by unilateral transection of the sciatic nerve. In the ipsilateral somatosensory cortex, as well as in the visual cortex at both cortical hemispheres, no significant changes in the cholinergic parameters studied could be detected. The data indicate that peripheral deafferentation of the somatosensory cortex results in a transient change of presynaptic cholinergic parameters within the affected somatosensory area as early as 1 to 3 days after the lesion; thus, they emphasize the involvement of cholinergic mechanisms in cortical reorganizational events.     

Chromatographic and enzymatic effects on transfer factor-like activity from human leukocytes and porcine spleen dialysate

1. The effect of dialysable transfer factor (TFd), derived from human leukocytes or porcine spleen cells, was measured using Listeria resistance in mice. 2. The molecular weight range of substance(s) containing TF-like activity is in the less than 3500 MW dialysis fraction on the basis of the capacity of peritoneal macrophages to produce superoxide anion (O2-). This biological activity is removed by heating at 56 degrees C. 3. After Sephadex G-10 chromatography of dialysates the significant activities are found in fractions III and IV of human leukocyte dialysate and in fractions of II and III of porcine spleen dialysate. 4. From enzymatic studies, most of the protective activity of both human leukocyte and porcine spleen dialysate is based on the action of small-molecular weight structures containing peptides and/or polynucleotides.     

Measurement of protein using bicinchoninic acid

Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.     

Production of monoclonal antibodies against avidin

Monoclonal antibodies of the IgG1 subclass were generated against chicken avidin. These antibodies were shown to be as sensitive as polyclonal antiserum in detecting avidin by radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) methods. Furthermore, the monoclonal antibodies were considerably more specific. Our results with a monoclonal anti-avidin RIA support previous findings that in inflammatory conditions avidin is synthesized also in other organs than the oviduct, although in the liver a major part of the activity detected by polyclonal anti-avidin RIA or biotin-bentonite assay was not due to avidin.     

Synthesis, biodistribution, and autoradiography of radiolabeled S-2-(3-methylaminopropylamino)ethylphosphorothioic acid (WR-3689)

35S- and 3H-labeled S-2-(3-methylaminopropylamino)ethylphosphorothioic acid (WR-3689) have been synthesized in our laboratory and used to study organ and cellular level distribution in C3H/Km mice bearing RIF-1 tumors. Tissue biodistributions obtained with 35S-WR-3689 showed that blood levels peak at 15 min postinjection and decline gradually over 60 min. At 30 min after drug injection the highest uptake is in kidney and submandibular salivary gland, with lowest levels in brain and moderate to low levels in the RIF-1 tumor, comparable to levels in skin and muscle. High resolution diffusible substance autoradiography with 3H-WR-3689 reveals a homogenous distribution of label over cells in liver and lung and nonuniform distribution of silver grains over the cytoplasm of cells in the kidney cortex, parotid and submandibular salivary glands, and small intestine. There are no indications of preferential nuclear location of label from protective drug in any tissue. Correlations of biodistribution and autoradiography data with measures of radioprotection in different tissues will be useful in interpreting mechanisms of radioprotection with this phosphorothioate.     

Growth-regulator Interactions in the Growth of the Shoot System of Avena sativa Seedlings: I. THE GROWTH OF THE FIRST INTERNODE

1. Segments, 3.5 mm. long, cut from the first internode of Avenasativa seedlings grown in complete darkness respond to bothauxins and gibberellic acid by accelerated extension. 2. The optimum concentration of indole-3-acetic acid (IAA) is10 p.p.m. and of gibberellic acid (GA) is 0.1 p.p.m. 3. The degree of stimulation relative to the growth of controlsegments is affected by the inclusion in the segement of thenode between the internode and coleoptile. Thus the gibberellineffect is greatly increased while the IAA effect is decreased.The optimal concentrations are not affected by inclusion ofthe node. 4. These results can best be explained in terms of the supplyby the node tissue of an endogenous auxin which is necessaryfor the expression of GA action. 5. Numerous factorial experiments demonstrated that there isno detectable interaction between applied IAA and GA in thepromotion of first-internode extension. This implies that thepostulated endogenous auxin which synergized GAA action in (4)is either an active form of IAA produced only in the node tissueor is a completely different auxin. 6. No synergism of growth-promotive action can be detected betweenGA and the two synthetic auxins I-naphthylacetic acid and 2,4-dichlorophenoxyaceticacid. 7. p-chlorophenoxy-iso-butyric acid (PCIB) anc 2,4,6-trichlorophenoxyaceticacid (2,4,6-T) act as weak auxins and thus antagonize competitivelythe promotive action of GA. 8. The anti-auxin -(I-naphythyl-methyl-sulphide)propionic acid(NMSP) antagonizes competitively the promotive action of bothIAA and GA. 9. The facts under (5)–(8) suggest that auxins and GAare acting at the same growth-promotion centres and may competefor them. 10. Growth inhibitions are induced by high concentrations ofPCIB, 2,4,6-T and NMSP. The inhibitions produced by PCIB and2,4,6-T are both synergized by supra-optimal concentrationsof IAA while that of NMSP is synergized by supra-optimal concentrationsof both IAA and GA. This similarity of the effects of IAA andGA suggests that their inhibition actions also are of a closelysimilar nature.     

Cytidine Analogues and Stomatogenic Recovery in Amicronucleate Paramecium tetraurelia and Paramecium jenningsi

Removal of the micronuclei of Paramecium tetraurelia and Paramecium jenningsi by micropipetting generates amicronucleate cell lines. These cell lines go through a period of growth depression for several dozen fissions, but they gradually recover. Amicronucleate cells in the depression period characteristically exhibit abnormal oral development, particularly reduction in the length of the buccal cavity and an abnormal pattern of the oral membranelles. To test the notion that the macronucleus is involved in the recovery of amicronucleate cell lines, DNA demethylation drugs were administered to amicronucleates in the depression period. After at least 4 fissions, the treated amicronucleates were assessed for their progress in recovery by scoring the proportion of cells with normal oral membranelles. Cvtidine analogues which demethylate cytosine specifically at the 5 position, namely 5-azacytidine, 5-aza-2'- deoxycytidine and 5-fluoro-2'-deoxycytidine. promoted recovery of the amicronucleates. Cytidine, 6-azacytidine, 2'-fluoro-2'-deoxy-cytidine and cytosine-β-D-arabinofuranoside did not. These results suggest that (i) 5-methylcytosine is present in the macronucleus of these Paramecium species, probably in small amounts and (ii) recovery of amicronucleates involves demethylation of macronuclear DNA. This implies that in normal cells the micronuclei are involved in maintaining the macronuclear DNA in a methylated state and hence the inactivation of the macronuclear sequences that are to be employed for stomatogenic recovery. A general mechanism for the control of gene expression may therefore be employed for the regulation of specific sequences.     

Radioprotection of normal tissues against gamma rays and cyclotron neutrons with WR-2721: LD50 studies and 35S-WR-2721 biodistribution

The ability of WR-2721 to protect mice against two modes of death following whole-body radiation with 137Cs gamma rays or d(22)+Be neutrons was examined. For single fractions, 400 mg/kg WR-2721 was administered prior to irradiation. In two-fraction exposures, the dose was 275 mg/kg given prior to each fraction. Dose modification factors (DMFs) were calculated as ratios of LD50 values. For single fractions of gamma rays, the DMF was 1.74 for the LD50/7 end point and for LD50/30, the DMF for single fractions was 2.25. For two fractions 3 hr apart, it was 1.88. For single fractions of cyclotron neutrons, the DMF was 1.32 for LD50/7. Measured with the LD50/30 end point, the DMF for single neutron doses was 1.41 and for two fractions, 1.19. Substantial radioprotection of bone marrow and intestinal epithelium against cyclotron neutrons was seen in these investigations. Biodistribution studies were done following ip injection of 35S-labeled WR-2721 into C3H mice bearing RIF-1 tumors. Blood levels peaked at 10 min after injection and declined thereafter. Most normal tissues achieved maximum levels of 35S at 30 to 60 min postinjection and high concentrations were retained in most tissues for up to 2 hr. Assuming that all 35S is in parent compound or dephosphorylated radioprotective metabolites, the concentration of protector (milligram per gram tissue) in various organs at 30 min postinjection ranked as follows: kidney greater than submandibular gland much greater than liver = lung greater than gut greater than heart much greater than blood greater than skin greater than tumor greater than brain. High levels of 35S were achieved and retention times were long in certain normal tissues which respond at early or late times postradiation and may be dose limiting in radiotherapy: kidney, liver, salivary gland, and lung. These combined observations suggest that there is potential for protecting dose-limiting, late-responding normal tissue in the radiotherapy of human cancer with both neutrons and conventional radiotherapy.