Ca2+ is Required by Clubroot Resistant Turnip Cells for Transient Increases in PAL Activity that Follow Inoculation with Plasmodiophora brassicae

Phenylalanine ammonia‐lyase (PAL, EC 4.3.1.5) activity in clubroot disease‐resistant turnip calli was transiently increased by 20 h after the inoculation with Plasmodiophora brassicae spores. The magnitude of the increase in PAL activity was four to six times higher than constitutive PAL activity. There was no transient increase in PAL activity in susceptible calli. Preincubation of calli in Ca²⁺‐free medium or the removal of Ca²⁺ from cell surfaces by ethylene glycol bis(2‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid‐chelation, completely inhibited induced PAL activity. The influx of exogenous Ca²⁺ into cells appears necessary for this pathogen induced PAL activity. Verapamil and the calmodulin inhibitor W7 almost completely inhibited induced PAL activity at 1 and 0.1 mm , respectively. Neomycin, ruthenium red and (1‐(6‐[(17β‐3‐Methoxyestra‐1,3,5‐(10)‐trien‐17‐yl)amino]hexyl)‐1H‐pyrrole‐2,5‐dione) did not inhibit induced PAL activity. Thus, verapamil and N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulphonamide hydrochloride‐sensitive Ca²⁺‐mediated signalling process appear necessary for P. brassicae induced PAL activity. As the protein synthesis inhibitor cycloheximide (CHX) blocked the induced increasing PAL activity, de novo synthesis of PAL appears to be required for turnip cell defence reactions against P. brassicae.     

Carbachol-induced reverse transformation of Chinese hamster ovary cells transfected with and expressing the m5 muscarinic acetylcholine receptor

Reverse transformation was induced in Chinese hamster ovary (CHO) cells transfected with and stably expressing the m5 subtype of the muscarinic acetylcholine receptor when stimulated with the muscarinic agonist, carbachol. Atropine, a muscarinic antagonist, blocked the carbachol-stimulated reverse transformation. CHO cells not transfected with the muscarinic receptor did not change with added carbachol. PMA induced reverse transformation without increasing cAMP accumulation in CHO cells. Carbachol, prostaglandin E2, and cholecystokinin increased cAMP accumulation but only carbachol caused reverse transformation. Carbachol-stimulated cAMP accumulation occurred at a higher concentration (EC50 10 microM) than did carbachol-stimulated reverse transformation (EC50 63 nM). Muscarinic m5 acetylcholine receptor transfected into CHO cells can induce reverse transformation which may be independent of cAMP.     

Modulation of cerebral acetylcholine metabolism by the dorsal diencephalic conduction system in the rat

We investigated the effects of interruption of the impulse flow in the habenulopeduncular pathways by local infusion of tetrodotoxin on the acetylcholine and choline content in selected dopamine rich regions in the forebrain and midbrain in rats. The tetrodotoxin infusion caused a marked increase in acetylcholine content in the medial frontal cortex, striatum and ventral tegmental area+interpeduncular nucleus, but not in the limbic area or the substantia nigra, whereas choline content was reduced only in both the striatum and ventral tegmental area+interpeduncular nucleus. There was an increase in 3,4-dihydroxyphenylacetic acid content in the striatum after the manipulation. These findings suggest that the dorsal diencephalic conduction system may be involved in the integration of the activity of cholinergic neurons in the forebrain and midbrain regions and striatal dopanine neurons may play a role in the modulation of cholinergic neurons.     

Detailed analysis of an amplified region at chromosome 11q13 in malignant tumors.

We have examined the amplification unit at chromosome band 11q13 in 12 primary tumors and one cancer cell line (A431) with 15 DNA markers. The amplified region and size varied from one tumor to another; the smallest amplicon was estimated to be 700 kb long and the largest was 4.5 Mb long at maximum, on the basis of a physical map. Furthermore, the DNA amplified in tumors was not always continuous, because in three cases one locus within the amplicon was not amplified. The amplified region common to all 13 cancers consisted of 500 kb of DNA which incorporated eight defined loci (BCL-1, cCI11-524, cCI11-283, cCI11-234, HBI-1, cCI11-454, HSTF1, and INT2). As two of them, cCI11-524 and cCI11-454, were found to contain DNA sequences conserved in other species, one or both of these loci might encode the gene(s) that may be associated with progression of these tumors.     

Identification of 57 conventional RFLP and 6 VNTR systems with 32 DNA clones on chromosome 11p15

Fifty-four clones containing human inserts were selected from a cosmid library constructed from a somatic cell hybrid containing chromosome 11p15.3-p15.5 as its only human complement. In 32 of these clones, 63 polymorphic systems were identified with a panel of restriction enzymes: 57 conventional RFLP systems and 6 highly polymorphic VNTR systems. Although we examined the cosmid with only seven enzymes, 18 clones (including 6 VNTRs) were polymorphic with three or more enzymes. The results suggested that DNA sequences on the peritelomeric region of chromosome 11p tend to be highly variable. Because these markers are highly informative, they will be excellent resources for investigations of hereditary diseases and tumor suppressor genes in this region of chromosome 11.     

Large deletions in the cytoplasmic kinase domain of the epidermal growth factor receptor do not affect its laternal mobility

The lateral diffusion coefficients of various epidermal growth factor (EGF) receptor mutants with increasing deletions in their carboxy-terminal cytoplasmic domain were compared. A full size cDNA construct of human EGF receptor and different deletion constructs were expressed in monkey COS cells. The EGF receptor mutants expressed on the cell surface of the COS cells were labeled with rhodamine-EGF, and the lateral diffusion coefficients of the labeled receptors were determined by the fluorescence photo-bleaching recovery method. The lateral mobilities of three deletion mutants, including a mutant that has only nine amino acids in the cytoplasmic domain, are all similar (D approximately equal to 1.5 X 10(-10) cm2/s) to the lateral mobility of the "wild-type" receptor, which possess 542 cytoplasmic domain of EGF receptor, including its intrinsic protein kinase activity and phosphorylation state, are not required for the restriction of its lateral mobility.     

α1-Adrenergic Receptor Mediates Arachidonic Acid Release in Spinal Cord Neurons Independent of Inositol Phospholipid Turnover

The alpha 1-adrenergic receptor has been shown to mediate the release of arachidonic acid in FRTL5 thyroid cells and MDCK kidney cells. In primary cultures of spinal cord cells, norepinephrine stimulated release of arachidonic acid (from neurons only) and turnover of inositol phospholipids (from neurons and glia) via alpha 1-adrenergic receptors. These two responses were dissociated by treatment with phorbol ester and pertussis toxin, which inhibited production of inositol phosphates with no appreciable effect on release of arachidonic acid. Extracellular calcium was required for release of arachidonic acid, but not for production of inositol phosphates. The calcium channel blockers nifedipine and verapamil inhibited release of arachidonic acid only. However, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a compound that blocks intracellular calcium release, diminished production of inositol phosphates, but had little effect on release of arachidonic acid. These results suggest that alpha 1-adrenergic receptors couple to release of arachidonic acid in primary cultures of spinal cord cells by a mechanism independent of activation of phospholipase C, possibly via the activation of phospholipase A2.