3D Triple-resonance NMR techniques for the sequential assignment of NH and 15N resonances in 15N- and 13C-labelled proteins

Summary Two new 3D ¹H-¹⁵N-¹³C triple-resonance experiments are presented which provide sequential cross peaks between the amide proton of one residue and the amide nitrogen of the preceding and succeeding residues or the amide proton of one residue and the amide proton of the preceding and succeeding residues, respectively. These experiments, which we term 3D-HN(CA)NNH and 3D-H(NCA)NNH, utilize an optimized magnetization transfer via the ²J_NC coupling to establish the sequential assignment of backbone NH and ¹⁵N resonances. In contrast to NH-NH connectivities observable in homonuclear NOESY spectra, the assignments from the 3D-H(NCA)NNH experiment are conformation independent to a first-order approximation. Thus the assignments obtained from these experiments can be used as either confirmation of assignments obtained from a conventional homonuclear approach or as an initial step in the analysis of backbone resonances according to Ikura et al. (1990) [Biochemistry, 29, 4659–4667]. Both techniques were applied to uniformly ¹⁵N- and ¹³C-labelled ribonuclease T1.     

A protocol for non-radioactive DNA labelling and detection in the RFLP analysis of rice and tomato using single-copy probes

An improved protocol for non-radioactive labelling and detection of rice and tomato DNA is described. The procedure includes the use of polymerase chain reaction for the incorporation of digoxigenin-dUTP in the DNA molecule and the use of a chemiluminescent compound (AMPPD) for the signal detection.     

Amino acid content in xylem sap of regrowing alfalfa (Medicago sativa L.): Relations with N uptake,N2 fixation and N remobilization

During vegetative regrowth of Medicago sativa L., soil N, symbiotically fixed N₂ and N reserves meet the nitrogen requirements for shoot regrowth. Experiments with nodulated or non-nodulated plants were carried out to investigate the changes in N flows originating from the different N sources and in xylem transport of amino acids during regrowth. Exogenous N uptake, N₂ fixation and endogenous N remobilization were estimated by ¹⁵N labelling and amino acids in xylem sap were analysed. Removal of shoots resulted in great declines of exogenous N flows derived either from N₂ or from NH₄NO₃ during the first week of regrowth, thereafter recovery increased linearly. Mineral N uptake as well as N₂ fixation occurred mainly between the 10th and 18th day after removal of shoots while exogenous N assimilation in intact plants remained at a steady level. Nitrogen remobilization rates in defoliated plants increased by at least three to five-fold, especially during the first 10 days following shoot removal. Compared to control plants, contents of amino acids in xylem sap, during the first 10 days of regrowth, were reduced by about 72% and 82% in NH₄NO₃ grown and in N₂ fixing plants, respectively. Asparagine was the main amino acid transported in xylem sap of both treated plants. Its relative contents during this period significantly decreased from 75% to 59% and from 67% to 36% respectively in non-nodulated plants and in nodulated ones. This decline was accompanied by compensatory increase in the relative contents of aspartate and glutamine.     

Subcellular localization of IpaC,an invasion plasmid antigen of Shigella dysenteriae type 1 and its in-vitro binding capability to mammalian cells

Invasion plasmid antigen C (IpaC), a 45-kDa protein encoded by an invasion plasmid of Shigella, is associated with the invasion of epithelial cells by the bacteria. Invasive strains of S. dysenteriae type 1 secreted more proteins into the extracellular environment than a non-invasive strain and secreted more IpaC protein. An anti-IpaC mouse monoclonal antibody was used as a probe to determine the subcellular localization of IpaC and its involvement in invasion of mammalian cells. Immunogold labelling of ultrathin sections of invasive bacteria indicated that the IpaC was only present in the cytoplasmic membrane and cytoplasm. There were no gold-IgG particles on the bacterial surface. Immunoblot analysis of different cellular fractions confirmed that the protein was associated with the inner cytoplasmic membrane and cytosolic fraction. The in-vitro binding capability of the IpaC protein was assessed using HeLa and isolated rat intestinal epithelial cells. The binding of the protein to the surface of mammalian cells indicates that it may have a role in the early stages of the infection process. The binding was sensitive to the action of proteolytic enzymes.     

Distribution of actin microfilaments in frog skin epithelial granular cells

Summary— Microfilaments were localised by immunofluorescence and immunogold cytochemistry to examine their distribution in granular cells of the isolated frog skin epithelium. Strongly fluorescent bundles of actin were observed beneath the plasma membrane with little evidence for actin in the central regions. Higher resolution offered by cytochemistry revealed that bundles of actin filaments comprised a substantial portion of the cortical cytoskeleton. Quantitative analysis of the frequency of gold label revealed an extremely rich array of filaments beneath the apical membrane of granular cells, with markedly less babel along the basolateral membrane and in the central cytoplasm. Treating cells with cytochalasin B or arginine vasopressin caused an apparent disruption of the apical actin fibres, concurrent with a decrease in gold label density. Assumably these signs are indicative of depolymerization of the filaments. Although the significance of this distribution is unknown, the apical polarisation of actin is consistent with a role in regulating the Na⁺ permeability of the apical membrane. The data are discussed in relation to possible roles of the cytoskeleton in the regulation of transepithelial sodium transport by vasopressin.     

Distribution of galanin-immunoreactive nerve fibers in the carotid labyrinth of the bullfrog,Rana catesbeiana: comparison with substance P-immunoreactive fibers

Immunoreactivity of galanin (GAL) was detected in the nerve fibers distributed within the intervascular stroma of the bullfrog carotid labyrinth. GAL-immunoreactive fibers are numerous, and some are close to the sinusoidal plexus. Most GAL fibers appear as thin processes with some varicosities. A combination of indirect double immunofluorescence labelling and image processing clearly demonstrated that the distribution patern of GAL fibers is different from that of SP fibers. This indicates that GAL and SP do not coexist in the same nerve fibers. The role of GAL fibers may be different from that of previously reported neuropeptides (substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, neuropeptide Y, and others) as a neuromodulator in controlling vascular tone of the labyrinth.     

Immunolocalization of pheromone-binding protein and general odorant-binding protein in olfactory sensilla of the silk moths Antheraea and Bombyx

The distribution of odorant-binding proteins among olfactory sensilla of three moth species was studied by immuno-electron microscopy. Two polyclonal antisera were used in a post-embedding labelling protocol on sections of cryo-substituted antennae. The first was directed against the pheromone-binding protein (PBP) of Antheraea polyphemus, the second against the general odorant-binding protein (GOBP) of the same species. Immunoblots showed that these antisera were highly specific; both antisera did, however, cross-react with related proteins in the related species A. pernyi, and in the bombycid moth B. mori. PBP and GOBP were localized only in olfactory sensilla trichodea and sensilla basiconica, the principal site being the sensillum lymph surrounding the sensory dendrites. In the males of all three species, the pheromone-sensitive long sensilla trichodea exclusively contained PBP. the majority of the sensilla basiconica in both sexes in these species contained GOBP; these sensilla are known to respond to plant and other general odours. Some sensilla were not labelled by either antiserum; presumably, these held an odorantbinding protein of a different subfamily. Never were PBP and GOBP co-localized in the same sensillum. Two observations deserve special attention: (1) PBP was also found in a few sensilla in females, and (2) in B. mori, where the long sensilla trichodea have a different functional specificity in males (pheromone) and females (plant odours), the expression of the odorant-binding protein (males: PBP; females: GOBP) is similarly different. The distinct and complex distribution pattern of odorant-binding proteins supports the notion that these proteins participate in stimulus recognition.Dedicated to Professor Ya.A. Vinnikov on the occasion of his 85. birthdayThis work was partly supported by DFG grant ste 501/3-1.     

电镜原位杂交观察小鼠中期染色体内RNA分布特点及其来源

本文用RNase-gold标记法处理小鼠中期染色体切片,发现染色单体切面内部及周边有大量较均匀分布的RNA。以玉米18 s rRNA的cDNA及水稻5srRNA的DNA为探针,经电镜原位杂交观察到了小鼠18srRNA和5srRNA均在中期染色单体切面内部及边缘有分布,从而首次证明小鼠中期染色体RNA除来源于核仁外,还可以来源于核基质中的5srRNA,但这并非仅有的两种来源。     

Part of the 23S RNA located in the 11S RNA fragment is a constituent of the ribosomal peptidyltransferase centre

Upon irradiation, 3-[4-benzoylphenyl]propionyl-PhetRNA bound to the P-site of poly(U)-primed ribosomes is exclusively cross-linked to 23S RNA. It is shown that the photoreaction only occurs with pyrimidine nucleotides. The site of the cross-link is located within an 11S RNA fragment, which comprises the 1100 nucleotides at the 3'-end of 23S RNA. The cross-linked Phe tRNA derivative is still functionally active in peptide bond formation. The site labelled on the 11S fragment is therefore an integral part of the peptidyltransferase centre.     

Binding of a spin-labelled photoallergen to human serum albumin

The binding site for 3,3',4',5-tetrachlorosalicylanilide (T4CS), a potent photoallergen, on human serum albumin (HSA) was studied by electron spin resonance spectroscopy using a spin-labelled analogue 3,5-dichlorosalicylamido-4-(2,2,6,6-tetramethylpiperidine 1-oxyl) (DCS-TEMPO) of T4CS in the absence of ultraviolet irradiation. DCS-TEMPO bound non-covalently (K = 5.8 X 10(6) M-1) to one major binding site on HSA. This binding site could be blocked by the photochemical binding of T4CS to the protein. Limited tryptic digestion of HSA or chemical modification of its single tryptophan residue with 2-hydroxy-5-nitrobenzyl bromide was found to reduce the binding constant of the T4CS/DCS-TEMPO-binding site. These observations are in good agreement with earlier conclusions on the nature of the T4CS-binding site and suggest a location for this site close to the single tryptophan residue of the HSA molecule.