Neuromedin U-immunoreactivity in the nervous system of the small intestine of the pig and its coexistence with substance P and CGRP

Summary In the small intestine of the pig, neuromedin U (NMU)-immunoreactivity was mainly confined to the nerve plexus of the inner submucosal and mucosal regions. After colchicine treatment, a high number of immunoreactive nerve cell bodies was observed in the plexus submucosus internus (Meissner), whereas only a low number was found in the plexus submucosus externus (Schabadasch). The plexus myentericus as well as the aganglionic nerve meshworks in the circular and longitudinal smooth muscle layers almost completely lacked NMU-immunoreactivity. Double-labeling experiments demonstrated the occurrence of distinct NMU-containing neuron populations in the plexus submucosus internus: (1) relatively large type-II neurons revealing immunoreactivity for NMU and calcitonin gene-related peptide (CGRP) and/or substance P (SP); (2) a group of small NMU- and SP-immunoreactive neurons; (3) a relatively low number of small neurons displaying immunoreactivity for NMU but not for SP. Based on its distributional pattern, it is concluded that NMU plays an important role in the regulation and control of mucosal functions.     

Selective adsorption/desorption of nucleic acids on submicron-sized polymeric particles

DNA can be removed or separated by the selective adsorption/desorption on positively charged submicronsized polymeric particles (SSPP). The selective adsorption of DNA, in the presence of protein, on positively charged SSPP was accomplished by increasing the concentration of potassium phosphate or sodium phosphate. The adsorption of DNA was not affected by the concentration of potassium phosphate or sodium phosphate up to 1.2M. On the other hand, the adsoprtion of a protein (bovine serum albumin) was completely impeded by 170mM potassium phosphate. DNA adsorbed on SSPP could be desorbed by increasing the concentration of NaCl or KCl, thus it can be recovered. DNA desorbed from SSPP when the concentration of NaCl or KC was higher than 0.6M. A complete desorption of DNA was achieved at the concentration of NaCl or KCl above 1.2M.     

Functional role of newly formed pore complexes in postmitotic nuclear reorganization

Many nuclear proteins are released into the cytoplasm at prometaphase and are transported back into the daughter nuclei at the end of mitosis. To determine the role of this reentry in nuclear remodelling during early interphase, we experimentally manipulated nuclear protein uptake in dividing cells. Recently we and others have shown that signal-dependent, pore complex-mediated uptake of nuclear protein is blocked in living cells on microinjection of the lectin wheat germ agglutinin (WGA), or of antibodies such as PI1 that are directed against WGA-binding pore complex glycoproteins. In the present study, we microinjected mitotic PtK₂ cells with WGA or antibody PI1 and followed nuclear reorganization of the daughter cells by immunofluorescence and electron microscopy. The inhibitory effect on nuclear protein uptake was monitored by co-injection of the karyophilic protein nucleoplasmin. When injected by itself early in mitosis, nucleoplasmin became sequestered into the daughter nuclei as they entered telophase. In contrast, nucleoplasmin was excluded from the daughter nuclei in the presence of WGA or antibody PI1. Although PtK₂ cells with blocked nuclear protein uptake completed cytokinesis, their nuclei showed a telophaselike organization characterized by highly condensed chromatin surrounded by a nuclear envelope containing a few pore complexes. These findings suggest that pore complexes become functional as early as telophase, in close coincidence with nuclear envelope reformation. They further indicate that the extensive structural rearrangement of the nucleus during the telophase-G1 transition is dependent on the influx of karyophilic proteins from the cytoplasm through the pore complexes, and is not due solely to chromosome-associated components.Abbreviations WGA wheat germ agglutinin - GlcNAc N-acetylglucosamine     

Molecular cloning of salicylate hydroxylase genes from Pseudomonas cepacia and Pseudomonas putida

The sal gene encoding Pseudomonas cepacia salicylate hydroxylase was cloned and the sal encoding Pseudomonas putida salicylate hydroxylase was subcloned into plasmid vector pRO2317 to generate recombinant plasmids pTK3 and pTK1, respectively. Both cloned genes were expressed in the host Pseudomonas aeruginosa PAO1. The parental strain can utilize catechol, a product of the salicylate hydroxylase-catalyzed reaction, but not salicylate as the sole carbon source for growth due to a natural deficiency of salicylate hydroxylase. The pTK1- or pTK3-transformed P. aeruginosa PAO1, however, can be grown on salicylate as the sole carbon source and exhibited activities for the cloned salicylate hydroxylase in crude cell lysates. In wild-type P. cepacia as well as in pTK1- or pTK3-transformed P. aeruginosa PAO1, the presence of glucose in addition to salicylate in media resulted in lower efficiencies of sal expression P. cepacia apparently can degrade salicylate via the meta cleavage pathway which, unlike the plasmid-encoded pathway in P. putida, appears to be encoded on chromosome. As revealed by DNA cross hybridizations, the P. cepacia hsd and ht genes showed significant homology with the corresponding plasmid-borne genes of P. putida but the P. cepacia sal was not homologous to the P. putida sal. Furthermore, polyclonal antibodies developed against purified P. cepacia salicylate hydroxylase inactivated the cloned P. cepacia salicylate hydroxylase but not the cloned P. putida salicylate hydroxylase in P. aeruginosa PAO1. It appears that P. cepacia and P. putida salicylate hydroxylases, being structurally distinct, were probably derived through convergent evolution.     

Comparison of effects of a potassium channel opener BRL34915, a specific potassium ionophore valinomycin and calcium channel blockers on endothelin-induced vascular contraction

The effects of a potassium (K+) channel opener BRL34915 and a specific K+ ionophore valinomycin on vasoconstriction induced by endothelin (ET) were compared with those of calcium (Ca2+) channel blockers, nicardipine and verapamil, using helical strips from rat thoracic aorta. ET induced potent and persistent contraction in control solution and similar but smaller contraction in Ca2+-free solution. BRL34915 and valinomycin inhibited the ET-induced contraction dose-dependently in control solution, but not in Ca2+-free solution. The ET-induced contraction was also inhibited by nicardipine and verapamil, though less strongly. On the other hand, high K+ (35 mM)-induced vasoconstriction was strongly inhibited by nicardipine and verapamil, but not by BRL34915 or valinomycin. These results support the idea that the extracellular Ca2+-dependent component of the ET-induced contraction may be mediated by Ca2+ influx by a route other than voltage-dependent Ca2+-channels.     

A dynamic model for the structure of acyl carrier protein in solution

The determination of solution structures of proteins using two-dimensional NMR data is commonly based on the assumption that the structure can be represented by a single rigid conformer. We present here a procedure whereby this assumption can be relaxed and illustrate its application to acyl carrier protein from Escherichia coli, a small negatively charged protein with no internal disulfide bonds. The methodology rests on a model having two distinct conformers in dynamic equilibrium. Use of this two-state model results in a dramatic improvement in fit to cross-relaxation-derived distance constraints and a substantial lowering of molecular mechanics energies for individual conformers of acyl carrier protein. The two-state model retains the three-helix motif previously identified on the basis of a one-state structure, but substantial motion of loop regions and the C-terminal peptide, as well as partial disruption of the second helix, is suggested to occur. Support for the existence of these motions can be found in amide exchange rate and spin relaxation time data.     

Effect of the hinge protein on the heme iron site of cytochrome c1

X-ray absorption spectroscopic (XAS) studies on cytochrome C1 from beef heart mitochondria were conducted to identify the effect of the hinge protein [Kim, C.H., & King, T.E. (1983) J. Biol. Chem. 258, 13543-13551] on the structure of the heme site in cytochrome c1. A comparison of XAS data of highly purified "one-band" and "two-band" cytochrome c1 [Kim, C.H., & King, T.E. (1987) Biochemistry 26, 1955-1961] demonstrates that the hinge protein exerts a rather pronounced effect on the heme environment of the cytochrome c1: a conformational change occurs within a radius of approximately 5 A from the heme iron in cytochrome c1 when the hinge protein is bound to cytochrome c1. This result may be correlated with the previous observations that the structure and reactivity of cytochrome c1 are affected by the hinge protein [Kim, C.H., & King, T.E. (1987) Biochemistry 26, 1955-1961; Kim, C.H., Balny, C., & King, T.E. (1987) J. Biol. Chem. 262, 8103-8108].     

Site-specificity of histone H1 methylation by two H1-specific protein-lysine N-methyltransferases from Euglena gracilis

1. The histone H1 fractions from rat spleen and liver were used as substrates for two H1-specific protein-lysine N-methyltransferases, V-A and V-B (protein methylase III) from Euglena gracilis. 2. When the enzymatically [methyl-3H]labeled H1 fractions were resolved by two-dimensional gel electrophoresis, four subtypes were found to be methylated (H1b, H1c, H1d and H1e). Both enzymes methylated H1c and H1b to approximately the same extent; H1d and H1e were methylated preferentially by enzyme V-B and V-A, respectively. 3. Histone H1c, [methyl-3H]labeled by the methyltransferase V-A, which had been digested by arginine-specific protease (Arg C protease), showed a single radioactive peptide on HPLC, indicating methylation site specificity of the enzyme. 4. Arg C protease-digestion of [methyl-3H]labeled H1c labeled by methyltransferase V-B indicated that this enzyme methylated two sites on the histone molecule. 5. The histone H1c methylation sites of these two enzymes did not overlap, indicating the two enzymes have different site specificity. 6. In combination with the other results, this suggests that the two enzymes serve discrete purposes, possibly involving the presumed different actions of histone H1 subtypes.     

Effect of enzymatic methylation of apocytochrome c on holocytochrome c formation and proteolysis

1. Methylation of the lysine at residue 72 of yeast apocytochrome c increases its import into mitochondria. 2. Using methylated and unmethylated apocytochrome c as substrate and intact yeast mitochondria and a solubilized mitochondrial fraction as a source of cytochrome c heme lyase, the results show that the methylation state of the apoprotein has no significant effect on its conversion to holoprotein. 3. The above result suggests that the import mechanism is separate from the heme-attaching activity. 4. Unmethylated apocytochrome c was less resistant to a yeast homogenate fraction that methylated apocytochrome c, suggesting that methylation of apocytochrome c alters the conformation of the whole protein.