CGRP immunoreactivity and NADPH-diaphorase in afferent nerves of the rat penis

Calcitonin gene-related peptide (CGRP)-immunoreactive afferent nerve fibers are abundant in the rat penis. In addition, NADPH-diaphorase, which stains for nitric oxide synthase, has been localized within both autonomic and sensory dorsal root ganglia (DRG) and may be part of an important biochemical pathway involved in penile tumescence. The purpose of this study was: 1) to examine the circuitry of afferent nerves that are CGRP immunoreactive from the L6 DRG, 2) to examine the possibility that there are NADPH-diaphorase-positive afferent fibers from the L6 DRG to the rat penis, and 3) to examine the localization and colocalization of CGRP and NADPH-diaphorase within L6 DRG afferent perikarya. Calcitonin gene-related peptide immunostaining in the penis was eliminated following a bilateral transection of the pudendal nerves, but was unchanged following a bilateral transection of the pelvic splanchnic or hypogastric nerves. The NADPH-diaphorase staining was not altered by any of the nerve transections. Injection of the retrograde axonal tracer fluorogold (FG) into the dorsum penis labeled perikarya in the L6 DRG. Although the majority of FG-labeled perikarya contained neither CGRP nor NADPH-diaphorase, small subpopulations of perikarya contained either CGRP immunoreactivity, NADPH-diaphorase, or both. A unilateral pudendal nerve transection virtually eliminated (>99%) FG labeling in the ipsilateral L6 DRG. These data suggest that NADPH-diaphorase and CGRP are present, either together or separately, within a subpopulation of penile afferent perikarya. In addition, CGRP-immunoreactive afferent nerve fibers reach the penis primarily via the pudendal nerves. Finally, NADPH-diaphorase-positive penile afferents may be another important source of nitric oxide (NO) for penile tumescence.     

Detection of the gangliosideN-glycolyl-neuraminyl-lactosyl-ceramide by biotinylatedEscherichia coli K99 lectin

K99 lectin fromEscherichia coli was purified and biotinylated via its carboxyl groups using biocytin hydrazide and a water soluble carbodiimide. Biotinylation of two out of the nine carboxyl groups was sufficient to permit detection of the lectin by avidin and did not cause any loss of the haemagglutinating activity. It was demonstrated that the biotinylated K99 lectin retained other important properties of native K99 and that it will probably become a very sensitive detecting reagent. Indeed, it was able to bind to HeLa cells, as do intact bacteria carrying K99 fimbriae, and also to recognizeN-glycolyl-neuraminyl-lactosyl-ceramide in an overlay binding assay. Abbreviations: NeuAc,N-acetylneuraminic acid; NeuGc,N-glycolylneuraminic acid; PBS, phosphate buffered saline (0.9% NaCl containing 150mm sodium phosphate, pH 7.2); LPS, lipopolysaccharide; BCHZ, biocytin hydrazide; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; BSA, bovine serum albumin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; DMEM, Dulbecco's modified Eagle medium. For the gangliosides, trivial names and structures are given according to the recommendations in [43]. NeuAc2-3Gal1-4Glc1-1Cer (NeuAc-GM₃); NeuGc2-3Gal1-4Glc1-1Cer (NeuGc-GM₃); GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer (GM₂); NeuAc2-8NeuAc2-3Gal1-4Glc1-1Cer (GD3); Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer (GM₁); NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-3)Gal1-4Glc1-1Cer (GD_1a); Gal1-3GalNAc1-4(NeuAc2-8NeuAc2-3)Gal1-4Gle1-1Cer (GD_1b); NeuAc2-3Gal1-3GalNAc1-4(NeuAc2-8NeuAc2-3) Gal1.-4Glc1-1Cer (GT_1b). NeuGc2-3Gal1-4GleNAc1-4Gal1-4Glc1-1Cer (NeuGc-SPG).     

Huge increase in bacterivores on freshly killed barley roots

Abstract Adding fresh roots to intact soil cores resulted in marked increases in microbial and microfaunal activity at the resource islands. Microbial activity increased in two phases following root addition. Respiratory activity and concentration of respiratory enzyme (dehydrogenase) in soil adhering to the roots was very high during the first three weeks resulting in anaerobic conditions in the soil. After a period of low respiratory activity and enzyme content, these quantities increased from 6 to 20 weeks, but not enough to maintain anaerobic conditions. Numbers of protozoa peaked earlier than the nematodes. Based on yield coefficients of microbes and bacterivores, the increase in bacterivores was in accordance with root-induced respiration activity. In soil adhering to roots, numbers of bacterial grazers (protozoa and nematodes) were up to 80 and 30 times higher, respectively, than in the surrounding soil. This effect is up to 20 times higher than observed around live root systems, which may suggest that the rhizosphere effect on microbivores could for the major part result from the decomposition of dead segments of the root system.     

Synthesis of the biologically active β-subunit of human nerve growth factor in Escherichia coli

The gene(NGFB) encoding the β subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters p_R and p_L, and expressed in Escherichia coli. The recombinant protein represented approximately 3% of the total cellular protein. Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule. Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons. These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E. coli.     

Gene expression in nematode-infected plant roots

Summary A major pathogen of potato plants (Solanum tuberosum) is the potato cyst nematode (Globodera spp.), which induces localized redifferentiation of a limited number of host cells to form a specialized feeding-site termed the syncytium. A novel strategy utilizing the polymerase chain reaction (PCR) was employed to construct a cDNA library from dissected potato roots highly enriched in syncytial material. The library was differentially screened with cDNA probes derived from the infected root tissue from a compatible interaction and from healthy root tissue. Characterization of one gene identified by the library screen indicated an expression pattern that correlated with events in the immediate vicinity of the pathogen after syncytial establishment. The strategy for library construction and screening could be applicable to the study of gene expression in any plant-pathogen interaction in which the limited supply of cells at the interface of the two organisms precludes a more traditional approach.     

Root hydrotropism of an agravitropic pea mutant, ageotropum

We have partially characterized root hydrotropism of an agravitropic pea mutant, ageotropum (from Pisum sativum L. cv. Weibull's Weitor), without interference of gravitropism. Lowering the atmospheric air humidity inhibited root elongation and caused root curvature toward the moisture-saturated substrate in ageotropum pea. Removal of root tips approximately 1.5 mm in length blocked the hydrotropic response. A computer-assisted image analysis showed that the hydrotropic curvature in the roots of ageotropum pea was chiefly due to a greater inhibition of elongation on the humid side than the dry side of the roots. Similarly, gravitropic curvature of Alaska pea roots resulted from inhibition of elongation on the lower side of the horizontally placed roots, while the upper side of the roots maintained a normal growth rate. Gravitropic bending of Alaska pea roots was apparent 30 min after stimulation, whereas differential growth as well as curvature in positive root hydrotropism of ageotropum pea became visible 4–5 h after the continuous hydrostimulation. Application of 2,3,5-triiodobenzoic acid or ethyleneglycol-bis-( β -aminoethylether)-N,N,N',N'-tetraacetic acid was inhibitory to both root hydrotropism of ageotropum pea and root gravitropism of Alaska pea. Some mutual response mechanism for both hydrotropism and gravitropism may exist in roots, although the stimulusperception mechanisms differ from one another.     

Inhibition of auxin-induced elongation and xyloglucan breakdown in azuki bean epicotyl segments by fucose-binding lectins

Auxin-induced elongation of epicotyl segments of azuki bean ( Vigna angularis Ohwi and Ohashi cv. Takara) was suppressed by fucose-binding lectins from Tetragonolobus purpureus Moench and Ulex europaeus L. These lectins also inhibited auxin-induced cell wall loosening (decrease in the minimum stress-relaxation time of the cell walls) of segments. Auxin caused a decrease in molecular mass of xyloglucans extracted with 24% KOH from the cell walls. The lectins inhibited auxin-induced changes in molecular mass of the xyloglucans. The autolytic release of xylose-containing products from the pectinase-treated cell walls was also suppressed by the lectins. Fucose-binding lectins pretreated with fucose exhibited little or no inhibitory effect on auxin-induced elongation, cell wall loosning, or breakdown of xyloglucans. These results support the view that the breakdown of xyloglucans is involved in the cell wall loosening responsible for auxin-induced elongation in dicotyledons.     

Ammonium and nitrate uptake by barley genotypes in diurnally fluctuating root temperatures simulating till and no-till conditions

The morphological development and N uptake patterns of spring barley (Hordeum vulgare L.) genotypes of Northern European (Nordic) and Pacific Northwest US (PNW) origin were compared under two diurnally fluctuating root temperature regimes in solution culture. The two regimes, 15/5°C and 9/5°C day maximum/night minimum temperatures, simulated soil temperature differences between tilled vs. heavy-residue, no-till conditions, respectively, observed during early spring in eastern Washington. Previous field experiments indicated that some of the Nordic genotypes accumulated more N and dry matter than the PNW cultivars during early spring under no-till conditions. The objective of this experiment was to determined whether these differences 1) are dependent on the temperature of the rooting environment, and 2) are correlated with genotypic differences in NH₄ ⁺ and NO₃ ^– uptake. Overall, shoot N and dry matter accumulation was reduced by 40% due to lower root temperatures during illumination. Leaf emergence was slowed by 14 to 22%, and tiller production was also inhibited. All genotypes absorbed more ammonium than nitrate from equimolar solutions, and the proportion of total N absorbed as NH₄ ⁺ was slightly higher in the 9/5°C than the 15/5°C regime. A Finnish genotype, HJA80201, accumulated significantly more shoot N than the PNW cultivars, Clark and Steptoe, and also more than a Swedish cultivar, Pernilla, in the 9/5°C regime. In the 15/5°C regime Steptoe did not differ in shoot N from the Nordic genotypes, while Clark remained significantly lower. These differences were not correlated to relative propensity for N form. Root lengths of the Nordic genotypes were significantly greater than the PNW genotypes grown under the 9/5°C regime, while the root lengths in the warmer root temperture regime were not significantly different among genotypes. Higher root elongation rates under low soil temperature conditions may be an inherent adaptive mechanism of the Nordic genotypes. Overall, the data indicate that lower maximum daytime temperatures of the soil surface layer likely account for a significant portion of the growth reductions and lower N uptake observed in no-till systems.     

Effective nodulation of rhamnaceous actinorhizal plants induced by air dry soils

Air dry soil samples stored at room temperature for more than one and a half years, were used as inocula for actinorhizal plants. Seedlings of Colletia paradoxa, Discaria americana, D. articulata and D. trinervis (Rhamnaceae) cultivated in nitrogen-free nutrient solution, were inoculated by adding dry soil to the solution. All the soil samples tested were able to induce nodulation, showing the presence of infective propagules of Frankia. Healthy growth of nodulated plants suggested the occurrence of nitrogen fixation.     

An inflatable minirhizotron system for root observations with improved soil/tube contact

Commonly used minirhizotrons consisting of a transparent tube inserted into the soil seldom attain good contact between the tube and the soil, which leads to root growth occurring in a gap rather than in the soil. A new system is described involving an inflatable flexible rubber wall, made from a modified motorcycle tube. Pressure ensures a proper tube/soil contact so that the environmental circumstances for root growth along the tube more closely correspond to those in the undisturbed soil. Before the endoscope slide is introduced into the minirhizotron for taking pictures, the inflatable tube is removed, so that there is no-often opaque-wall between the endoscope and the roots. This improves the picture quality and facilitates the analysis of root images.