Effect of serotonergic agonists in the nucleus accumbens on d-amphetamine-stimulated locomotion.

Serotonergic projections from the raphe nuclei are thought to modulate locomotor activity in the rat, and serotonin injection into the nucleus accumbens attenuates the hypermotility elicited by amphetamine. The purpose of the present study was to characterize the effects of various classes of serotonergic agonists administered into the nucleus accumbens on d-amphetamine-stimulated locomotor activity in order to determine which serotonin receptor subtypes are involved. Administration of the nonselective 5-HT agonist quipazine, the 5-HT-1 agonist mCPP, the 5-HT-1a agonist 8-OH-DPAT, the 5-HT-1b agonist CGS-12066B, and the 5HT-1c/2 agonist DOI did not inhibit d-amphetamine-stimulated locomotor activity. Pronounced lateral head weaving was noted after 8-OH-DPAT administration. The combination of the 5-HT-1a agonist 8-OH-DPAT and the 5-HT-1b agonist CGS-12066B, however, did inhibit d-amphetamine-stimulated locomotor activity. In contrast, the 5-HT-3 agonist 1-phenylbiguanide enhanced the locomotor effect of d-amphetamine. This effect was partially reversed by the 5-HT-3 antagonist MDL-7222. These studies suggest that serotonin has complex and multiple effects on the regulation of locomotor activity within the nucleus accumbens.     

Glucocorticoid-induced gastric mucosal damage: inhibition of leukotriene, but not prostaglandin biosynthesis

The effects of daily administration of ulcerogenic doses of the glucocorticoids, dexamethasone and prednisolone, on gastric prostaglandin and leukotriene synthesis were examined in the rat. Significant gastric damage was not observed until the fourth day of treatment with dexamethasone and until the sixth day of treatment with prednisolone. However, the onset of gastric damage was not accompanied by any significant effect of these drugs on gastric 6-keto prostaglandin Fl alpha synthesis. Conversely, both drugs caused a reduction in gastric leukotriene C4 synthesis, with dexamethasone producing a highly significant (p less than 0.001) effect. Furthermore, there was a highly significant (p less than 0.001) correlation between the ability of these drugs to inhibit gastric leukotriene C4 synthesis, and their ability to reduce gastric tissue levels of the neutrophilic enzyme myeloperoxidase. Thus, glucocorticoid-induced gastric damage does not appear to be related to inhibitory effects of these drugs on prostaglandin synthesis. Whether or not effects of these drugs on gastric leukotriene C4 synthesis and myeloperoxidase activity are relevant to the mechanism of ulceration is unclear.     

Effects of clipping and soil compaction on growth,morphology and mycorrhizal colonization of Schizachyrium scoparium,a C4 bunchgrass

Summary A factorial design of clipping and compaction was used to study the responses of Schizachyrium scoparium and its mycorrhizal symbionts to these stresses. All treatment combinations significantly reduced the growth and biomass of plants relative to controls. Compaction significantly reduced tillering and crown expansion while clipping increased tillering early in the growing season and reduced it later. Mycorrhizal colonization of roots was highest in the clipped plots and lowest in compacted plots. Spore number was highest in compacted plots and lowest in clipped plots. It appears that spore number may be negatively correlated with root growth since any treatment that reduced plant growth yielded higher spore numbers. The combination of clipping and compaction reduced plant growth the most, but had intermediate effects on mycorrhizal colonization and spore number.     

C-terminal bombesin sequence requirements for binding and effects on protein synthesis in Swiss 3T3 cells.

The active-site serine of the extracellular beta-lactamases of Streptomyces cacaoi and Streptomyces albus G has been labelled with beta-iodopenicillanate. The determination of the sequence of the labelled peptides obtained after trypsin digestion of the denatured proteins indicate both enzymes to be class A beta-lactamases. Surprisingly the two Streptomyces enzymes do not appear to be especially homologous, and none of them exhibited a high degree of homology with the Streptomyces R61 DD-peptidase. Our data confirm that, as a family of homologous enzymes, class A is rather heterogeneous, with only a small number of conserved residues in all members of the class.     

Semisynthesis of cytochrome c analogues. The effect of modifying the conserved residues 38 and 39.

We have used chemical and enzymic protein engineering techniques to create analogues of the semisynthetic two-fragment complex (1-37).(38-104) of mitochondrial cytochrome c. This complex, unlike the natural product of specific tryptic cleavage, (1-38).(39-104), from which it is prepared, quite closely resembles the parent protein in functional characteristics and is thus a suitable substrate for modifications designed to study structure-function relations. We have replaced the invariant Arg-38 and the conserved Lys-39 with a range of alternative amino acids and have studied the effects on the principal functional parameters. The hydrogen-bonding capacity of Arg-38 is crucial to the stabilization of the bottom omega-loop, while the positive charge of Lys-39 helps maintain the high redox potential by electrostatic effects at the haem iron.     

Functional consequences of the excision of an omega loop, residues 40-55, from mitochondrial cytochrome c

A novel technique for protein semisynthesis, enzymic activation, has been used to create a mitochondrial cytochrome c analogue in which the conventional bottom loop has been deleted. The resulting structure resembles that of cytochrome c555 from a primitive photosynthetic sulfur bacteria. Comparisons of this analogue with natural cytochromes show which of the functional differences between cytochromes c and c555 may be related directly to the incorporation of the loop. The structure is an example of an omega loop, recently defined as a discrete category of protein secondary structure. The analogue maintains the overall structure of the parent protein, but a significant change in redox potential has been engineered. It provides support for the prediction that omega loops act as independent modules in folding, function, and evolution. The rapidity of the synthesis and the high yield of product show that this technique for protein engineering is both competitive with, and complementary to, genetic methods.     

Development of a single probe for documentation of chimerism following bone marrow transplantation.

Although numerous genetic markers are available for studying chimerism after bone marrow transplantation (BMT), there remains a need for a practical and highly informative method that is applicable in the early posttransplantation period. Using DNA restriction-fragment-length polymorphisms (RFLPs), we have evaluated the feasibility of developing a single synthetic oligonucleotide probe to study post-BMT chimerism. We have thus tested three candidate probes, termed O-3315-32, O-3315-80, and O-AY-29, that are homologous to tandemly repetitive sequences. Our results demonstrated donor-specific and recipient-specific fragments in 11 of 11 HLA-matched sibling pairs tested using probes O-3315-32 and O-3315-80. When probe O-AY-29 was used, 14 of 17 sibling pairs showed both donor and recipient markers, one had only a recipient marker, and two were identical. We showed that each of the three synthetic probes was effective in documenting donor marrow engraftment, mixed hematopoietic chimerism, the patient's pre-BMT phenotype (by using cultured skin fibroblasts obtained after BMT), and the origin of the malignant hematopoietic cells (i.e., of donor or recipient origin) in patients who developed recurrent hematologic malignancy following BMT. Compared with the use of cloned genomic probes, there are several important advantages to the use of synthetic oligonucleotide probes in studying post-BMT chimerism. Synthetic probes have absolute hybridization specificity and can be designed to suit the purposes of an individual study, since they have adjustable specificity that can be altered by changes in the length of the probe and by changes in the hybridization temperature. A single synthetic probe analogous to several highly polymorphic loci can have a polymorphism information content sufficiently high so that all but a small percentage of BMT patients could be followed easily; for example, if a probe were complementary to three highly polymorphic unlinked loci, it would discriminate approximately 98% of sibling donor/recipient pairs. This would be accomplished using only one restriction-endonuclease digestion and only one gel electrophoresis. Since other genetic markers, e.g., red blood cell antigens, immunoglobulin allotypes, and chromosome analysis, are not uniformly informative and, in some cases, cannot be used in the early posttransplantation period, the use of synthetic oligonucleotide probes for analysis of DNA RFLP is emerging as the method of choice for studies of post-BMT chimerism. This method will allow for the development of new knowledge that has not been possible with previous methods.     

The bioflavonoid quercetin inhibits neutrophil degranulation, superoxide production, and the phosphorylation of specific neutrophil proteins

Quercetin, a C-kinase antagonist, inhibits neutrophil degranulation and superoxide production induced by f-met-leu-phe, solid phase IgG, zymosan treated serum and a phorbol ester (PMA). Quercetin is more effective in inhibiting degranulation (IC50 = 20 uM) than superoxide production (IC50 = 80 microM). Neutrophil activation by PMA is accompanied by the phosphorylation of neutrophil proteins of 205, 170, 130, 91, 77, 67, 56, 47, 39, 34, 27, and 20 kilodaltons; quercetin also inhibits the phosphorylation of these proteins. Dose-response studies indicated that phosphorylation of the 67 kilodalton protein was particularly sensitive to inhibition by quercetin at concentrations that also inhibit neutrophil degranulation and superoxide production. These results suggest that phosphorylation of the 67 kilodalton protein may be an important intracellular reaction associated with neutrophil activation.     

Phosphorylation of nucleolin by a nucleolar type NII protein kinase

Nucleolin [C23 or 100 kilodaltons (kDa)] is the major nucleolar phosphorylated protein in exponentially growing Chinese hamster ovary cells. A nucleolar cyclic nucleotide independent protein kinase copurified with nucleolin in a complex which could be dissociated by hydroxyapatite chromatography. The kinase was stimulated by spermine and inhibited by heparin and presented most of the properties of nuclear casein kinase NII. Kinetic analyses showed the apparent Km value for nucleolin (7 X 10(-4) mg/mL) to be lower than those for other casein kinase II substrates such as nuclear protein HMG 14 (0.15 mg/mL), topoisomerase I (0.025 mg/mL), or topoisomerase II (0.04 mg/mL). Similarly, Vmax values were higher for nucleolin than for other substrates. Nucleolin thus appears to be a natural preferential substrate of nucleolar casein kinase NII. The kinase phosphorylated nucleolin in vitro at serine residues in a 29-kDa CNBr fragment located near the amino terminus of the molecule. The enzyme labeled typical casein kinase II sites. These sites were found predominantly in two highly acidic tryptic fragments designated A (residues 21-49) and C (residues 180-221) which contained serines having at least two acidic residues on their carboxyl-terminal sides. These results demonstrate the existence in the nucleolus of a type of NII protein kinase that uses a protein involved in ribosome assembly as preferential substrate.     

Synthesis of dihydrothymidine and thymidine glycol 5'-triphosphates and their ability to serve as substrates for Escherichia coli DNA polymerase I

5,6-Dihydrothymidine 5'-triphosphate (DHdTTP) was synthesized by catalytic hydrogenation of thymidine 5'-triphosphate (dTTP). Thymidine glycol 5'-triphosphate (dTTP-GLY) was prepared by bromination of dTTP followed by treatment with Ag2O. The modified nucleotides were extensively purified by anion-exchange high-performance liquid chromatography (HPLC). Alkaline phosphatase digestion of DHdTTP and dTTP-GLY gave the expected products (5,6-dihydrothymidine and cis-thymidine glycol), the identities of which were confirmed by reverse-phase HPLC using authentic markers. HPLC analysis of the alkaline phosphatase digested DHdTTP revealed that DHdTTP was a mixture of C5 diastereoisomers [(5S)- and (5R)-DHdTTP]. Despite the significant distortion of the pyrimidine ring in DHdTTP, it was incorporated in place of dTTP during primer elongation catalyzed by Escherichia coli DNA polymerase I Klenow fragment. The rate of incorporation of DHdTTP was about 10-25-fold lower than that of dTTP. On the other hand, dTTP-GLY, which also has a distorted pyrimidine ring, did not replace dTTP, and no elongation of the primer was observed. In order to study the preference of incorporation of the diastereoisomers of DHdTTP into DNA, salmon testes DNA, activated by exonuclease III, was used as a template for DNA polymerase I Klenow fragment in the presence of [3H]DHdTTP (S and R mixture) and normal nucleotides. After enzymatic digestion of the DNA to nucleosides, the products were analyzed by HPLC. The ratio of the isomers incorporated into DNA (S:R = 73.27) was virtually the same as that of the [3H]DHdTTP substrates (S:R = 79.21).(ABSTRACT TRUNCATED AT 250 WORDS)