刺激隐神经中C类纤维诱发的小脑浦肯野细胞简单...

Simple spike of cerebellar Purkinje cells (PC-SS) was recorded with microelectrode. In the NCCVF (normalized cross-covariance function) histogram, spontaneous PC-SS does not show obvious peak. When the saphenous nerve is stimulated at lower intensities, which elicits the A-fiber input only, the discharge response (A-CED) consists of an early component with a latency of 16.7 +/- 0.9 ms and a late component with a latency of 270.8 +/- 12.8 ms. After A-fibers are blocked selectively by polarizing current, the stimulation at a suprathreshold strength for C-fiber evokes a characteristic response (C-CED) with a latency of 142.4 +/- 4.3 ms. However, the C-CED can not be evoked by the inputs of A- and C-fiber simultaneously. In NPSDF histogram, the spontaneous activities of PC-SS can be divided into two groups, the high and the low peak group. The high peak group (n = 15) has a peak energy value of 15.7 +/- 4.7 x 10(-3) and peak frequency of 4.07 +/- 1.69 Hz. A-fiber input causes an increase of the peak value, while C-fiber input causes a decrease. The low peak group (n = 16) has a peak energy value 8.4 +/- 1.4 x 10(-3) and peak frequency of 3.67 +/- 2.90 Hz. Both A-fiber and C-fiber inputs cause an increase of the peak value, but the effect of A-fiber input was more prominent. The results show that the pure C-fiber input can reach the cerebellar PC and elicit characteristic simple spike response.     

降香黄檀着叶期和无叶期次生韧皮部筛分子的超微结构

利用透射电镜技术研究了生长在海南岛的热带落叶树降香黄檀(Dalbegia odorifera T.Chen)1—2年生枝条着叶期和无叶期次生韧皮部筛分子的超微结构,并就这两个时期的筛分子进行了比较。着叶期每个成熟筛分子内有一个带尾的纺锤形P-蛋白质体,主体由稠密而散乱的P-蛋白质细纤维组成,尾部呈结晶状;筛分子具有横向端壁和单筛板,在邻近筛板处,细胞壁向筛分子腔内形成明显的突起。无叶期仍然保持着与着叶期大致相同厚度的有功能韧皮部,筛分子具有正常的原生质体,P-蛋白质和筛板孔的结构也与着叶期的相同,但筛分子内有较多的淀粉粒和囊泡。     

Transgenic turf-type tall fescue (Festuca amndinacea Schreb.) plants regenerated from protoplasts

To improve turfgrasses using genetic engineering, we have developed a transformation system in turf-type tall fescue, one of the most important turfgrass species. Embryogenic cell cultures were established after callus induction from embryos of mature seed. The agarose-bead method with nurse cells was used to culture protoplasts and plants were regenerated from protoplasts of tall fescue cultured cells. To develop transgenic tall fescue plants, the hygromycin resistance gene and the -glucuronidase gene were introduced into the tall fescue protoplasts by electroporation. A high concentration (200 mg/l) of hygromycin was required to select transformed cells because of the high level of endogenous resistance to the antibiotic in tall fescue. Most of the transformed cells exhibited GUS activity and several plants were regenerated from these cells. The presence of introduced genes was confirmed by Southern blot hybridization of PCR amplified DNA from transgenic plants.Abbreviations Adh alcohol dehydrogenase - BAP benzylaminopurine - bp base pair(s) - GUS -glucuronidase - Kb kilobase(s) - MS Murashige and Skoog's medium - PCR polymerase chain reaction     

Replacement of lysine-181 by aspartic acid in the third transmembrane region of endothelin type B receptor reduces its affinity to endothelin peptides and sarafotoxin 6c without affecting G protein coupling.

A conserved aspartic acid residue in the third transmembrane region of many of the G protein-coupled receptors has been shown to play a role in ligand binding. In the case of endothelin receptors, however, a lysine residue replaces this conserved aspartic acid residue. To access the importance of this residue in ligand binding, we have replaced it with an aspartic acid in the rat endothelin type B (ETb) receptor by PCR mediated mutagenesis. The binding characteristics and functional properties of both the wild type and mutant receptors were determined in COS-7 cells transiently expressing the cloned receptor cDNAs. Using 125I-ET-1 as the radioactive peptide ligand in displacement binding studies, the wild type receptor displayed a typical non-isopeptide-selective binding profile with similar IC50 values (0.2-0.6 nM) for all three endothelin peptides (ET-1, ET-2, and ET-3) and sarafotoxin 6c (SRTX 6c). Interestingly, the mutant receptor showed an increase in IC50 values for ET-1 (5 nM), ET-2 (27 nM), and ET-3 (127 nM) but displayed a much larger increase in IC50 value for SRTX 6c (> 10 uM). The lysine mutant receptor still elicited full inositol phosphate (IP) turnover responses in the presence of saturating concentrations of endothelins (10 nM of ET-1, 100 nM of ET-2, or 1 uM of ET-3), indicating that the mutation (K181D) did not affect the coupling of mutant receptor to the appropriate G protein. These results demonstrate that lysine-181 on the receptor is important for binding ET peptides; however, it is required for binding the ETb selective agonist-SRTX 6c.     

Calcium-activated potassium channels expressed from cloned complementary DNAs.

Calcium-activated potassium channels were expressed in Xenopus oocytes by injection of RNA transcribed in vitro from complementary DNAs derived from the slo locus of Drosophila melanogaster. Many cDNAs were found that encode closely related proteins of about 1200 aa. The predicted sequences of these proteins differ by the substitution of blocks of amino acids at five identified positions within the putative intracellular region between residues 327 and 797. Excised inside-out membrane patches showed potassium channel openings only with micromolar calcium present at the cytoplasmic side; activity increased steeply both with depolarization and with increasing calcium concentration. The single-channel conductance was 126 pS with symmetrical potassium concentrations. The mean open time of the channels was clearly different for channels having different substituent blocks of amino acids. The results suggest that alternative splicing gives rise to a large family of functionally diverse, calcium-activated potassium channels.     

Production of hepatitis delta virus and suppression of helper hepatitis B virus in a human hepatoma cell line.

The hepatitis delta virus (HDV) is a defective virus with a coat composing of the surface antigen of its helper virus, hepatitis B virus (HBV). Replication of HDV in the absence of HBV has been shown in cell cultures by transient transfection of the HDV plasmid. However, the formation and release of HDV virions have not been observed. In this report, a human hepatoma cell line HuH-7 was transiently cotransfected with HDV and HBV plasmids. The production of monomeric and multimeric antigenomic RNAs of HDV in the transfected cells indicated replication of the HDV genome. The major 3.5- and 2.1-kb RNAs of HBV were also expressed. Virions of both HDV and HBV were released from the cotransfected cells, as shown by the detection of monomeric genomic HDV RNA and partially double-stranded HBV DNA in the culture medium. Thus, this is the first report that describes the assembly and the release of HDV viral particles in an in vitro cell culture. The HDV virions released possessed physicochemical properties identical to those of the HDV virions found in infected human serum. Furthermore, expression of both the 3.5- and 2.1-kb RNAs of HBV was shown to be dramatically decreased by the presence of HDV, indicating suppression of the expression of HBV genes by HDV. The amount of HBV virions released was similarly suppressed by HDV. Cotransfection of HBV with an expression plasmid of the HDV delta antigen remarkably reduced the levels of the 3.5- and 2.1-kb HBV RNAs, indicating that suppression of the expression of HBV RNAs by HDV occurs via the action of the delta antigen. This HBV- and HDV-cotransfected human hepatoma cell line should provide an excellent system for the study of the function of the delta antigen and the interaction between HDV and its helper, HBV.     

Disturbed nonlinear multispecies models in ecology.

We analyze a disturbed form of the general Lotka-Volterra model of an ecosystem with m interacting species. The disturbances act on the intrinsic growth rates of the species and are assumed to be bounded but otherwise unknown. We employ a Lyapunov technique and the concept of "reachable set" from control theory to estimate the set of all possible population densities that are attainable as a result of the disturbances. To calculate estimates for this reachable set, a number of numerical methods that entail the solution to one or more global optimization problems are developed. Specific examples involving two, three, and four species are solved. We also derive an explicit analytical expression that represents an estimate for the reachable set in the m-dimensional case. The estimate is conservative but can be evaluated without carrying out any optimization procedure. We show that methods developed in this paper can be applied to certain other types of nonlinear ecosystem models.     

Immunocytochemical Localization of Mandelonitrile Lyase in Mature Black Cherry (Prunus serotina Ehrh.) Seeds

Mandelonitrile lyase (MDL, EC 4.1.2.10), which catalyzes the reversible dissociation of (R)-(+)-mandelonitrile to benzaldehyde and hydrogen cyanide, was purified to apparent homogeneity from mature black cherry (Prunus serotina Ehrh.) seeds by conventional protein purification techniques. This flavoprotein is monomeric with a subunit molecular mass of 57 kilodaltons. Glycoprotein character was shown by its binding to the affinity matrix concanavalin A-Sepharose 4B with subsequent elution by α-methyl-d-glucoside. Upon chemical deglycosylation by trifluoromethanesulfonic acid, the molecular mass was reduced to 50.9 kilodaltons. Two-dimensional gel analysis of deglycosylated MDL revealed the presence of several subunit isoforms of similar molecular mass but differing slightly in isoelectric point. Polyclonal antibodies were raised in New Zealand white rabbits against deglycosylated and untreated MDL. Antibody titers were determined by enzyme linked immunosorbent and dot immunobinding assays, while their specificities were assessed by Western immunoblot analysis. Antibodies raised against untreated lyase recognized several proteins in addition to MDL. In contrast, antisera raised against deglycosylated MDL were monospecific and were utilized for developmental and immunocytochemical localization studies. SDS-PAGE and immunoblotting analysis of seed proteins during fruit maturation showed that MDL first appeared in seeds shortly after cotyledons began development. In cotyledon cells of mature seeds, MDL was localized primarily in the cell wall with lesser amounts in the protein bodies, whereas in endosperm cells, this labeling pattern was reversed. N-terminal sequence data was gathered for future molecular approaches to the question of MDL microheterogeneity.