Fluorescence-microscopical demonstration of a population of gastro-intestinal nerve fibres with a selective affinity for Quinacrine

Summary A population of nerve fibres in the gastro-intestinal tract of mice showing a high affinity for quinacrine was revealed by fluorescence microscopy. Similar results were obtained in rats and guinea pigs. Whole-mounts of sheets of the smooth muscle layer following incubation in 10^-6-10^-7 M quinacrine for 15–60 min revealed fine fluorescent varicose nerve fibers in the myenteric plexus of Auerbach both around nerve cell bodies and in the interconnecting strands. Many fibers were also present between the strands of the plexus, especially running parallel to the circular muscle layer. Such fibers were not seen in similarly quinacrine-incubated irides. A proportion of the cell bodies in Auerbach's plexus also showed quinacrine accumulation. These cells were apparently smaller neurons, sometimes with fluorescent processes. Intraperitoneal injections of quinacrine failed to demonstrate nerve fibers, but some cell bodies in Auerbach's plexus were positive. Subsequent paraformaldehyde treatment for monoamine visualization showed persistent adrenergic nerve terminals in the intestine and iris. These nerves seemed to be fewer and had a more yellow fluorescence than normally. The identity of the quinacrine-positive fibers is discussed with respect to recent suggestions that purinergic, substance P, enkephalin, and somatosin-containing nerves, in addition to adrenergic and cholinergic nerves, are present in the gut wall.Supported by the Swedish Medical Research Council (04X-03185). Magnus Bergvalls Stiftelse and Karolinska Institutets Fonder. For generous gifts of Mepacrine we thank Winthrop, Skärholmen, Stockholm, Sweden. The skilful technical assistance of Miss Gerd Boetius and Miss Maud Eriksson is gratefully acknowledged     

Formation of a functional adrenergic input to intraocular cerebellar grafts: Ingrowth of inhibitory sympathetic fibers

The intraocular transplantation technique was used to study the ingrowth of peripheral sympathetic adrenergic nerves from the iris into transplants of fetal rat cerebellum, and the possible function of these nerves. The transplants, grown in oculo for one-half to eight months, were analyzed by fluorescence histochemistry and electrophysiological techniques. Peripheral sympathetic adrenergic fibers from the iris were able to grow into the cerebellar transplants and arborize in a pattern similar to that in situ, appearing in all three cortical layers and the noncortical areas of the transplants. The density of visible nerves without pretreatment and after preincubation in 10⁻⁶ or 10⁻⁵ M α-methylnorepinephrine was comparable to mature rat cerebellum. The spontaneous discharge of the Purkinje cells in oculo was inhibited by microiontophoresis of norepinephrine (NE) and amphetamine in sympathetically innervated, as well as sympathectomized transplants denervated by ganglionectomy. The NE response was blocked by the adrenergic β-receptor blocker MJ-1999. GABA also inhibited the Purkinje cell activity while glutamate accelerated the discharge. Parenteral amphetamine inhibited Purkinje cell activity in sympathetically innervated transplants, but was ineffective in denervated transplants. The Purkinje cell spontaneous activity was inhibited by electrical stimulation of the NE fiber input through the cervical sympathetic trunk. This inhibition could be antagonized by parenteral reserpine or the β-adrenergic antagonist propranolol. The responses of the Purkinje cells within the transplants to drugs and transmitters mimic those of the adult rat in situ. In view of the fluorescence histochemical evidence for an ingrowth of peripheral sympathetic adrenergic fibers into the cerebellar transplants, and the results of stimulating the sympathetic trunk, it is suggested that peripheral adrenergic fibers may be able to establish functional connections with the Purkinje cells similar to the cerebellar adrenergic synapses normally formed in situ by fibers from the locus coeruleus.     

Triploidy—Observations in 154 Diandric Cases

Hydatidiform moles (HMs) are abnormal human pregnancies with vesicular chorionic villi, imposing two clinical challenges; miscarriage and a risk of gestational trophoblastic neoplasia (GTN). The parental type of most HMs are either diandric diploid (PP) or diandric triploid (PPM). We consecutively collected 154 triploid or near-triploid samples from conceptuses with vesicular chorionic villi. We used analysis of DNA markers and/or methylation sensitive-MLPA and collected data from registries and patients records. We performed whole genome SNP analysis of one case of twinning (PP+PM).In all 154 triploids or near-triploids we found two different paternal contributions to the genome (P1P2M). The ratios between the sex chromosomal constitutions XXX, XXY, and XYY were 5.7: 6.9: 1.0. No cases of GTN were observed. Our results corroborate that all triploid human conceptuses with vesicular chorionic villi have the parental type P1P2M. The sex chromosomal ratios suggest approximately equal frequencies of meiosis I and meiosis II errors with selection against the XYY conceptuses or a combination of dispermy, non-disjunction in meiosis I and meiosis II and selection against XYY conceptuses. Although single cases of GTN after a triploid HM have been reported, the results of this study combined with data from previous prospective studies estimate the risk of GTN after a triploid mole to 0% (95% CI: 0–1,4%).     

Chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum

The chemical modification of cysteine and tyrosine residues in formyltetrahydrofolate synthetase from Clostridium thermoaceticum has been examined relative to enzymatic activity and reactivity of these groups in the native protein. 4,4′-Dipyridyl disulfide, dansylaziridine, and fluorescein mercuric acetate all reacted with just one of six sulfhydryls per enzyme subunit, resulting in activities of 100, 95 and 70%, respectively. The K_m values for MgATP, formate, and tetrahydrofolate were unaltered in the modified enzymes. ATP did produce a 2.5-fold reduction in the rate of reaction between the enzyme and 4,4′-dipyridyl disulfide. Tetranitromethane reacted most rapidly with a single sulfhydryl group per subunit to produce a 20–30% loss in activity. Subsequent additions of tetranitromethane modified 2.2 tyrosines per subunit which was proportional to the loss of the remaining enzymatic activity. Folic acid, a competitive inhibitor, protected against modification of the tyrosines and the associated activity losses; however, the oxidation of the single sulfhydryl group and the initial 20–30% activity loss were unaffected. In the presence of folic acid, higher concentrations of tetranitromethane produced a loss of the remaining activity proportional to the modification of 1.2 tyrosines per subunit. It is proposed that at least 1 tyrosine critical for enzymatic activity is located at or near the folic acid/tetrahydrofolate binding site.     

Atomic interactions in protein-carbohydrate complexes. Tryptophan residues in the periplasmic maltodextrin receptor for active transport and chemotaxis.

We have refined the 1.9 A resolution crystal structures of two maltodextrin receptor mutants in which tryptophan residues 230 and 232 have been changed to alanine and compared these structures with the refined 1.7 A structure of the wild-type protein. In the wild-type structure, Trp230, which is located in the maltodextrin-binding groove, stacks against the B-face of the reducing sugar of the bound maltose. Trp232, which is located near the protein surface, does not participate directly in sugar binding. Relative to the wild-type structure, neither mutation caused a significant rearrangement in the overall protein structure or in the mode of binding maltose. Although the position once occupied by Trp230 remains empty, a new water molecule has moved near the void. In contrast, a new water molecule has entered into the space once occupied by Trp232. Whereas one hydrogen bond is formed with the water molecule near the Trp230 void, no hydrogen bond is associated with the water molecule occupying the space vacated by Trp232. The three van der Waals' contacts between Trp230 and maltose in the wild-type structure that are lost in the W230A mutation could contribute to the 12-fold decrease in ligand-binding activity of the mutant protein. The W232A mutation causes little change in binding activity. The structures of these mutant proteins also provide some insight into the complicated tryptophan fluorescence spectra of the maltodextrin binding-protein. The change in fluorescence due to the deletion of Trp230 can readily be explained as resulting directly from loss of Trp230 in the sugar-binding site. The change in fluorescence due to deletion of Trp232, however, is ascribed to the modification of local interactions mediated by the binding of maltodextrin since the tryptophan is not directly involved in any sugar-binding interaction.     

Water and Sulphate Uptake at Root Reduction in Ricinus communis

The aim of the investigation was to study the influence of the rate of water uptake on the uptake of sulphate at supernormal rates of water flow. This was achieved by reducing the size of the root system of 42 days old Ricinus plants. The rate of water flow through the root increased 3 times by reducing the root system to 20 percent. This did not change the retention of sulphate in the roots. The uptake of sulphate was proportional to the size of the root system and thus independent of the rate of water flow while the water uptake (transpiration) was a function of the size of the shoot and the resistance of the root. This was contrary to the conditions at a moderate rate of water flow, when water and sulphate uptake followed each other. The results are discussed in terms of the salt uptake as a series of active and passive processes.