A highly sensitive northern blot assay detects multiple proenkephalin a-like mRNAs in human caudate nucleus and pheochromocytoma

Total RNA from post mortem human caudate nucleus, cerebellum, cerebral cortex and pheochromocytoma tissues has been prepared. Northern blot analysis, using a single-stranded human proenkephalin A antisense probe (cRNA), revealed the existence of two different proenkephalin A-like sequences in the human caudate nucleus and pheochromocytoma RNA extracts of approximately 1400 and 1000 nucleotides in length respectively, whereas no specific RNA bands could be detected in the cortex and only the 1400 nucleotide band was present in the cerebellum. Under highly stringent hybridization conditions, the proenkephalin A-like RNA bands still appear, indicating that the detected RNA species have either identical or a closely related sequence to that of the wellcharacterized human proenkephalin A mRNA sequence.     

应用电子探针对植物根际和根内营养元素微区分布的探讨

用电子探针可检测出玉米、大豆根际和根内含有Na,Mg,Al,Si,P,S,Cl,K,Ca,Ti,Fe,Cu和Zn 13种元素。这些元素在根际土壤、粘液层和根组织内的含量分布有一定的规律性。除Si,Al,Ca,Fe在根际土壤中峰值较高外,Ti仅在土壤中达到可检测量;S,Fe和Zn富集在粘液层,Mg,P,Cl只在根组织内才有较明显的峰。这些规律可作为区分根—土界面的参考指标。K含量在根内明显高于根际土壤,并由表皮层到中柱径向增加;Ca则与K不同,且受植物种类的影响。     

单纯疱疹病毒分子生物学分型方法的研究

联合运用HSV-1和HSV-2型特异性核酸探针对28株HSV临床分离株做了鉴定分型,并与应用HSV型特异性单克隆抗体的三种免疫学方法的检测结果进行了比较。核酸探针与单克隆抗体之间的符合率为100%,但分型率不同,核酸探针为100%,单克隆抗体为64—82%。研究结果表明,本实验所建立的HSV-McAb-APAAP桥联免疫酶染技术具有简便、敏感、实用的特点。     

31P NMR analysis of intracellular pH of Swiss mouse 3T3 cells: Effects of extracellular Na+ and K+ and mitogenic stimulation

Summary Swiss mouse 3T3 cells grown on microcarrier beads were superfused with electrolyte solution during continuous NMR analysis. Conventional³¹P and¹⁹F probes of intracellular pH (pH _c ) were found to be impracticable. Cells were therefore superfused with 1 to 4mm 2-deoxyglucose, producing a large intracellular, pH-sensitive signal of 2-deoxyglucose phosphate (2DGP). The intracellular incorporation of 2DGP inhibited the Embden-Meyerhof pathway. However, intracellular ATP was at least in part retained and the cellular responsivity to changes in extracellular ionic composition and to the application of growth factors proved intact. Transient replacement of external Na⁺ with choline or K⁺ reversibly acidified the intracellular fluids. Quiescent cells and mitogenically stimulated cells displayed the same dependence of shifts in pH _c on external Na⁺ concentration (c _Na ^o ). pH _c also depended on intracellular Na⁺ concentration (c _Na ^o ). Increasingc _Na ^c by withdrawing external K⁺ (thereby inhibiting the Na,K-pump) caused reversible intracellular acidification; subsequently reducingc _Na ^o produced a larger acid shift in pH _c than with external K⁺ present. Comparison of separate preparations indicated that pH _c was higher in stimulated than in quiescent cells. Transient administration of mitogens also reversibly alkalinized quiescent cells studied continuously. This study documents the feasibility of monitoring pH _c of Swiss mouse 3T3 cells using³¹P NMR analysis of 2DGP. The results support the concept of a Na/H antiport operative in these cells, both in quiescence and after mitogenic stimulation. The data document by an independent technique that cytoplasmic alkalinization is an early event in mitogenesis, and that full activity of the Embden-Meyerhof pathway is not required for the expression of this event.     

Spin probe clustering in human erythrocyte ghosts

Summary A model has been developed for 5-nitroxide stearate, I(12,3), distribution in human erythrocyte ghosts which accurately predicts ESR spectral alterations observed with increased probe/total lipid (P/L) at 37°C. This spin probe occupies a class of high-affinity, noninteracting sites at low loading. Saturation occurs with increasing probe concentration, and, at higher loading, the probe inserts itself at initially dilute sites to form membranebound clusters of variable size. No low probe remains at high P/L where all I(12,3) clusters in a concentrated phase. This model allows determination of the dilute/clustered probe ratio, and shows that I(12,3) segregates in erythrocytes at what might otherwise be considered low P/L (e.g., 1/359). These findings validate the earlier use of empirical parameters to estimate probe sequestration in biological membranes.     

Studies on 9-amino-6-chloro-2-methoxy acridine fluorescence quenching and enhancement in relation to energy transduction in spheroplasts and intact cells of the cyanobacterium Plectonema boryanum

The fluorescent probe 9-amino-6-chloro-2-methoxy acridine was used to study the energy transduction in the thylakoid and cell membranes of the cyanobacterium Plectonema boryanum. Apart from light-driven electron transfer, the dark endogenous respiration also leads to energization resulting in an ACMA fluorescence response, that is sensitive to the electron flow inhibitor 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone, to the energy transfer inhibitors dicyclohexylcarbodiimide and venturicidine and to the uncoupler 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide.In spheroplasts, in which the cell membranes have lost their capacity to maintain a proton gradient, the respiration-and light-induced ACMA fluorescence changes (quenching) are similar to those in chloroplasts. In intact cells a combination of reversible quenching and enhancement of ACMA fluorescence was found. This dualistic behaviour is supposedly caused by an opposite orientation of the thylakoid and cell membranes. ACMA quenching at the level of the thylakoids was obtained either by respiratory or photosynthetic electron transfer and gave similar responses to those obtained in the spheroplasts. The slower ACMA fluorescence enhancement, only observed in cells with intact cell membranes, also evoked by both respiration and light-induced energization is sensitive to the compounds mentioned above and in addition to KCN.Our results support the view [8] that dark oxidation of substrates by O₂ proceeds via the thylakoid membrane and terminates at a CN^- sensitive oxidase located in the cell membrane which requires the involvement of a mobile cytoplasmic redox mediator.Abbreviations ACMA 9-amino-6-chloro-2-methoxy acridine - chl a chlorophyll a - DBMIB 2, 5-dibromo-3-methyl-6-isopropyl-p-benzoquinone - DCCD dicyclohexylcarbodiimide - DNP dinitrophenol - DNP-INT dinitrophenyl ether of 2-iodo-4-nitrothymol - FCCP carbonylcyanide-p-trifluoro-methoxy phenylhydrazone - S-13 5-chloro-3-t-butyl-2-chloro-4-nitrosalicylanilide - tricine N-2 (2-Hydroxy-1, 1-bis (hydroxymethyl) ethyl)-glycine - Tris Tris (hydroxymethyl) amino methane     

Insulin uptake by rat liver endothelium studied in fractionated liver cell suspensions

Summary Cellular distribution of insulin receptors was studied in fractionated rat liver cell suspensions using ¹²⁵1-insulin and a visual probe consisting of latex beads covalently linked to insulin (minibeads). Fractionation was done on metrizamide gradients which yielded two cellular fractions. The large cell fraction consisted mostly of hepatocytes and the small cell fraction consisted of 37% endothelial cells as well as Kupffer cells. The magnitude of insulin uptake by the endothelium-rich small cell fraction was at least double that of the uptake by the hepatocyte-rich fraction. The minibead technique demonstrated that in the small cell fraction only endothelial cells, and not Kupffer cells, were responsible for the insulin uptake. Our findings suggest that liver endothelium may be responsible for the uptake of circulating insulin and its transport to hepatocyte. This emphasizes the presence of a tissue-blood barrier in the liver.Abbreviations PRS phosphate-buffered saline - SEM scanning electron microscopy - TEM transmission electron microscopy     

Molecular characterization and genetic mapping of two clusters of genes encoding chlorophyll a/b-binding proteins in Lycopersicon esculentum (tomato)

We have constructed a tomato genomic library in the λ Charon 4 phage vector. The library was screened with a pea cDNA probe encoding a chlorophyll a/b-binding protein (CAB), and several recombinant phages containing tomato CAB genes were isolated and characterized by restriction mapping, heteroduplex analysis and nucleotide sequencing. Two phages with overlapping segments of the tomato genome contain a total of four CAB genes, all arranged in tandem. A third phage contains three CAB genes, two arranged in tandem and one in opposite orientation, and an additional, truncated CAB gene. Genetic mapping experiments showed that the four CAB genes on the first two phages belong to a locus, previously designated Cab-1, on chromosome 2. The CAB genes from the third phage belong to the Cab-3 locus on chromosome 3. Complete sequence determination of two CAB genes, one from each locus, and additional sequence determination of about 50% of each of the other five CAB genes showed that each gene within a CAB locus is more similar to other CAB genes in the same locus than it is to the CAB genes from the second locus. Furthermore, the polypeptides encoded by Cab-1 genes diverge significantly from those encoded by Cab-3 genes in the domains of transit peptide and the N terminus of the mature polypeptide but are essentially identical in the rest of the sequence.     

Nucleic acid hybridisation with a probe specific for 3 aminoglycoside acetyltransferase type IV: A survey of resistance to apramycin and gentamicin in animal strains of Escherichia coli

Abstract Resistance to apramycin due to production of a 3-aminoglycoside acetyltransferase type IV (AAC(3)IV) has recently been detected among Gram-negative bacteria isolated in France from bovine clinical samples. 24 apramycin-resistant Escherichia coli strains isolated over the country, and epidemiologically unrelated, were studied by colony hybridization using an intragenic probe specific for AAC(3)IV. The results obtained indicated that the structural gene for the acetyltransferase was present in all the isolates tested and in the corresponding apramycin-resistant transconjugants. This observation demonstrates that resistance to apramycin by acetylation of the antibiotic has spread very rapidly in bovine Gram-negative bacteria.     

Photochemical changes of fluorescent probes in membranes and their effect on the observed fluorescence anisotropy values

The fluorescence intensity of diphenylhexatriene (DPH) and of trimethylammonium-diphenylhexatriene (TMA-DPH) is measured when these probes are embedded in vesicles of dipalmitoyl- and dioleoylphosphatidylcholine (DPPC and DOPC), in mixtures of these vesicles as well as in vesicles of the mixed phospholipids, in trout intestinal brush border membranes and in mitoplasts of rat liver cells. The intensity in DOPC vesicles is found to be significantly higher than in DPPC vesicles. When these systems are irradiated with strong ultraviolet light radiation, a decrease in the fluorescence intensity is observed; this effect is much stronger in DOPC than in DPPC vesicles. The fluorescence anisotropy values in the mixture of vesicles as well as in the membranes show an initial increase with irradiation which is followed by a significant decrease. A transfer of DPH molecules between DPPC and DOPC vesicles is observed. For TMA-DPH this transfer takes place only from DPPC to DOPC vesicles, but not vice-versa. These results are related to intensity and anisotropy measurements of these probes in cell cultures.