Measurement of extracellular 5-HT and 5-HIAA in hippocampus of freely moving rats using microdialysis: long-term applications

Extracellular levels of 5-hydroxytryptamine (5-HT) and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were measured in the ventral hippocampus of the awake rat using microdialysis. The basal level of 5-HT in hippocampal dialysates was very close to the detection limit of our assay. However, addition of a 5-HT re-uptake blocker, citalopram, to the perfusion medium resulted in a 3-fold rise of 5-HT levels in dialysates and provided a stable baseline. Under these conditions, extracellular levels of 5-HT and 5-HIAA could be measured up to 11 days after dialysis probe implantation. 5-HT measured in dialysate was Ca²⁺-dependent both 24 h and 7 days after surgery. In comparison K⁺-induced depolarization caused a 9-fold increase in 5-HT output 24 h after probe implantation and this effect had disappeared 10 days later, although behavioral activation following K⁺ application was similar 24 h and 11 days after surgery. Systematic administration of -trytophan had no significant effect on 5-HT but increased 5-HIAA levels by 90%. These results suggest that in the presence of citalopram, 5-HT in hippocampal dialysates is derived from serotonergic neurones. The microdialysis method appears most useful for measurement of extracellular 5-HT 24–72 h after probe implantation. Finally, our data indicate that extracellular 5-HIAA mainly reflects intraneuronal metabolism of 5-HT prior to release.     

A method of isolation of apical membranous sheets from frog urinary bladder epithelium by stripping with gelatin

We have developed a technique for recovering apical membranous sheets from amphibian urinary bladders by gelatin stripping. The tissue is mounted on a lucite support and the apical surface is first stuck onto a gelatin-coated glass slide at 30 degrees C. This sandwich is then chilled on ice and the bladder is pulled away from the slide. Preliminary results indicate that this simple technique could be used to remove membranous apical sheets of various sizes, almost devoid of cytoplasmic contamination and without significant damage to the underlying cell structures. The method could also be adapted to prepare perforated cells and to study the cohesive forces between the different layers of the tissue.     

普通小麦栽培品种丰抗13的4B—1D染色体易位的鉴定

通过细胞学观察,在普通小麦栽培品种“丰抗13”和“京红1号”的杂交后代中,发现有多价体出现,这就表明有染色体易位发生。为进一步弄清究竟是哪条染色体发生了易位,我们采用单体测交方法,观察鉴定所有各单体系F_1的花粉母细胞第一次减数分裂中期Ⅰ(以下简称PMCs中Ⅰ)染色体构型。从鉴定结果发现,凡2n=42的F_1 PMCs中Ⅰ出现19~Ⅱ 1~Ⅳ,而2n=41的F_1PMCs中Ⅰ的染色体构型不同,单体与易位有关的两个单体系4B和1D F_1 PMCs中的Ⅰ构型中有部分呈现为19个二价体加1个三价体,即19~Ⅱ 1~Ⅲ,没有单价体,而其余各单体系F_1 PMCs中Ⅰ构型则表现为18个二价体,1个四价体和1个单价体,即18~Ⅱ 1~Ⅰ 1~Ⅳ。因此,可以肯定“丰抗13”存在1个染色体易位,其有关染色体就是4B和1D。     

Ca2+ channels in chick neural retina cells characterized by 1,4-dihydropyridine antagonists and activators

The voltage-sensitive calcium channel in cultured chick neural retina cells was characterized by the actions of the enantiomers of Bay K 8644 and 202-791 and other 1,4-dihydropyridines. These cells showed time- and voltage-dependent Ca2+ uptake that was stimulated by K+ depolarization and blocked by the inorganic calcium channel blockers Cd2+ and Co2+. A small fraction only (15% maximum) of the uptake was inactivated by predepolarization of the cells with 80 mM K+. Ca2+ uptake was sensitive to the 1,4-dihydropyridine calcium channel antagonists and activators. (S)-Bay K 8644 and (S)-202-791 stimulated the Ca2+ uptake, and (R)-Bay K 8644 and (R)-202-791 as well as nitrendipine and PN 200-110 inhibited Ca2+ uptake stimulated by K+ depolarization or channel activators. The K+ depolarization-stimulated uptake was inhibited by 90%, but the activator-stimulated uptake was completely blocked by the 1,4-dihydropyridine antagonists. The potencies of these agents as inhibitors of Ca2+ uptake were significantly lower than the binding affinities in membrane preparations from the same cells or their binding and pharmacologic affinities in vascular smooth muscle. K+ depolarization or (S)-Bay K 8644 induced 45Ca2+ uptake was not observed in a glial cell culture. [3H]Nitrendipine and [3H]PN 200-110 bound to membrane preparations of the cells consistent with the presence of a single type of high affinity binding site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anaerobic dechlorination of 2,4-dichlorophenol in freshwater sediments in the presence of sulfate.

In the presence of added sulfate, 2,4-dichlorophenol and 4-chlorophenol were transformed stoichiometrically to 4-chlorophenol and phenol, respectively, in anaerobic freshwater lake sediments between 18 and 40 degrees C. The concomitantly occurring sulfate reduction reduced the initial sulfate concentration from 25 mM to about 6 to 8 mM and depressed methane formation.     

A general pre-steady-state solution to complex kinetic mechanisms

We have developed a general method for solving transient kinetic equations using Laplace transforms. Laplace transforms can be used to transform systems of differential equations that describe pre-steady-state kinetics to systems of linear algebraic equations. The general form of the pre-steady-state solution is (formula; see text) where I(t) is the time dependence of the physically observed property of the system, n is the number of intermediates, lambda i are the observed rate constants (reciprocals of the relaxation times), t is time, and Ii are the amplitude coefficients associated with each observed rate constant. We have written a program in compiled BASIC to run on a personal computer to evaluate Ii and lambda i. The program will evaluate the rate constants and coefficients of a mechanism with eight intermediates and seven relaxation times in 4 s on an 8-MHz PC-XT equipped with a math coprocessor. The most complex mechanism that we have solved, a mechanism containing 20 intermediates and 19 relaxation times, required approximately 5 min. We believe that this method will be useful to evaluate the differences in transient properties of complex biochemical mechanisms.     

Low nanogram detection of nucleotides using fast atom bombardment-mass spectrometry

The effect of trimethylsilyl (TMS) derivatization on detection limits of mononucleotides in fast atom bombardment-mass spectrometry (FAB-MS) was examined. FAB-MS methods were developed to optimize sensitivity using adenosine 5'-monophosphate as a model compound and then applied to reference standards of two clinically important nucleotides: tricyclic nucleoside-5'-monophosphate (TCNMP) and 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP). The detection limit for the TMS derivative of TCNMP was 2.5-5 ng/microliters and less than 2.5 ng/microliters for FdUMP as its TMS derivative. This is greater than two orders of magnitude more sensitive than the FAB-MS analysis of the corresponding free compounds. These low detection limits for the TMS derivatives were obtained using a narrow scan range, signal averaging, detection in the negative ion mode, and 3-nitrobenzyl alcohol as the matrix. Hydrolysis of one or more of the labile TMS groups did occur, with the extent of hydrolysis being greatest in the more protic matrices.     

Polyclonal antibodies to rabbit skeletal muscle protein phosphatases C-I and C-II

Polyclonal antibodies against rabbit skeletal muscle phosphatases C-I and C-II were raised in goats and in mice. The goat polyclonal antibodies to phosphatases C-I and C-II were examined for their ability to immunoblot the purified enzymes and crude rabbit muscle extracts. In preparations of phosphatases C-I and C-II that were apparently homogeneous, the expected ca. 35- to 38-kDa polypeptides were immunoblotted, but, in addition, immunoblotting of a 67-kDa polypeptide was observed. Both the antisera blotted only the 67-kDa polypeptide in crude rabbit muscle extracts and not the expected 35- to 38-kDa polypeptides. These findings are qualitatively similar to those reported previously (D.L. Brautigan et al. (1985) J. Biol. Chem. 260, 4295-4305) where immunoblotting experiments with a sheep antisera to phosphatase C-I indicated that the ca. 35-kDa polypeptide originates from a 70-kDa precursor. On further investigation, it was found that our antisera were strongly immunoreactive to rabbit serum albumin. The antisera blotted purified rabbit albumin, but not bovine serum albumin. After passage through a rabbit albumin-Sepharose column, the antisera lost immunoreactivity to rabbit albumin, and no longer blotted the ca. 70-kDa band in muscle extracts or in purified enzyme preparations. These findings show that the phosphatase preparations contained traces of albumin which produced a strong antigenic reaction. Production of antisera in BALB/c mice produced similar results; i.e., an antibody to the low-molecular-weight phosphatases was produced that was also a strong antibody to rabbit albumin. This antibody could be removed by affinity adsoption on rabbit albumin-Sepharose columns. In addition, the antibodies to phosphatase C-I displayed no cross-reactivity to phosphatase C-II, while antibodies to C-II showed no cross-reactivity to phosphatase C-I by immunoblotting methods.     

Functional cysteinyl residues in human placental aldose reductase

Incubation of human placental aldose reductase (EC 1.1.1.21) with the sulfhydryl oxidizing reagents 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) and N-ethylmaleimide (NEM) results in a biexponential loss of catalytic activity. Inactivation by DTNB or NEM is prevented by saturating concentrations of NADPH. ATP-ribose offers partial protection against inactivation by DTNB, whereas NADP, nicotinamide mononucleotide (NMN), and the substrates glyceraldehyde and glucose offer little or no protection. The inactivation by DTNB was reversed by dithiothreitol and partially by 2-mercaptoethanol but not by KCN. When the release of 2-nitro-5-mercaptobenzoic acid was measured, 3 mol of sulfhydryl residues was found to be modified per mole of the enzyme by DTNB. Correlation of the fractional activity remaining with the extent of modification by the statistical method of C.-L. Tsou (1962, Sci. Sin. 11, 1535-1558) indicates that of the three reactive residues, one reacts at a faster rate than the other two, and that two residues are essential for the catalytic activity of the enzyme. Labeling of the total sulfhydryl by [14C]NEM and quantification of DTNB-reactive residues in the enzyme denatured by 6 M urea indicates that a total of seven sulfhydryl residues are present in the protein. The modification of the enzyme did not affect Km glyceraldehyde, but the modified enzyme had a lower Km NADPH. Kinetic analysis of the data suggests that a biexponential nature of inactivation could be due to the formation of a dissociable E:DTNB complex and the presence of a partially active enzyme species.