Construction of Chemical Libraries of Volatile Compounds by Combinatorial Synthesis of Homologous Mixtures: Alk-4-en-1-ols,Alk-4-enals and Methyl Alk-4-enoates

A compound library of sixty six linear compounds, eleven representatives of six molecular families: (E)- and (Z)-isomers of alk-4-en-1-ols, alk-4-enals, and methyl alk-4-enoates, was prepared by combinatorial syntheses to allow the creation of a mass spectral database directly usable for their identification in GC/MS analyses. We demonstrate here that compound libraries can be prepared by combinatorial syntheses using long linear synthetic sequences, i. e., eight step in the case of 4-enals. The resulting mixtures of homologues are still perfectly exploitable to deliver the requested information such as clean mass spectra and good gas chromatographic retention indices.     

Identification and quantitative determination of 3-chloro-2-hydroxypropylmercapturic acid and α-chlorohydrin in urine of rats treated with epichlorohydrin

Epichlorohydrin (ECH) is used in many industrial processes. Different toxic effects of ECH were found in rodents. The metabolism of ECH was investigated before in rats using [¹⁴C]ECH. The aim of this investigation was the development of non-radioactive quantitative analytical methods for measuring two urinary metabolites of ECH, namely 3-chloro-2-hydroxypropylmercapturic acid (CHPMA) and α-chlorohydrin (α-CH). The identity of CHPMA and α-CH excreted in urine of rats treated with 5 to 35 mg/kg ECH was confirmed by GC-MS. The quantitative analysis of CHPMA, involving ethyl acetate extraction from acidified urine and subsequent methylation and analysis by gas chromatography-flame photometric detection (GC-FPD), showed a method limit of detection of 2 μg/ml. The analysis of α-CH, based on ethyl acetate extraction and subsequent analysis by GC-ECD, showed a method limit of detection of 2 μg/ml. CHPMA and α-CH derivatives could be determined quantitatively down to concentrations of 0.5 and 0.4 μg/ml urine, respectively, by selected-ion monitoring GC-MS under EI conditions. Cumulative urinary excretion of CHPMA and α-CH by rats treated with ECH were found to be 31 ± 10 and 1.4 ± 0.6% (n = 13) of the ECH dose, respectively. For CHPMA, the dose-excretion relationship suggested partially saturated ECH metabolism. For α-CH, the dose-excretion relationship was linear. With fractionated urine collection it was found that approximately 74 and 84% of the total cumulative excretion of CHPMA and α-CH, respectively, took place within the first 6 h after administration of ECH. From these investigations it is concluded that the GC-FPD and GC-ECD based methods developed are sufficiently sensitive to measure urinary excretion of CHPMA and α-CH in urine from rats administered 5 to 35 mg/kg ECH. It is anticipated that the analysis of CHPMA and α-CH based on GC-MS may be sufficiently sensitive to investigate urinary excretion from humans occupationally exposed to ECH.     

人胸腺素α原cDNA的克隆及在大肠杆菌中的表达

采用基因工程方法制取人胸腺素α原获得成功。用２０ｕｇ／ｍｌ植物血球凝集素（ＰＨＡ）和５００Ｕ／ｍｌ重组人白细胞介素２（ＩＬ－２）联合刺激人胎儿胸腺细胞,从中提取总ＲＮＡ,经反转录ＰＣＲ获得了人胸腺素α原ｃＤＮＡ;将之克隆入ｐＵＣ１９中,序列测定表明与已报道序列一致,进一步将之亚克隆入原核表达载体ｐＢＶ２２０,转化大肠杆菌ＤＨ５ａ．观察到在不改变氨基酸编码的前提下,增加胸腺素ａ原上游引物中Ａ、Ｔ含量,可以明显提高胸腺素α原的表达量,同时,不同培养基对它的表达也有影响。胸腺素α原在大肠杆菌中以可溶形式表达,不需复性。初步活性测定显示,它可明显刺激人外周血淋巴细胞Ｅ－玫瑰花结形成率。重组人胸腺素α原在大肠杆菌中表达,为其临床应用及基础研究奠定了基础。     

Identification and partial characterization of α2-macroglobulin from the tuatara (Sphenodon punctatus)

α₂-Macroglobulin (α₂-M), a large molecular mass proteinase-binding protein, was identified in plasma from tuatara (Sphenodon), a rare reptile endemic to New Zealand. In this genus, α₂-M constitutes 11–13% of total plasma protein (∼2.2–3.9 mg/ml). Analysis of blood samples collected at approximately monthly intervals from individual tuatara indicated that the plasma level of α₂-M remains fairly constant. The subunits of tuatara α₂-M have an apparent molecular mass of ∼160 kDa as determined by SDS-polyacrylamide gel electrophoresis and the intact protein is an oligomer that contains inter-chain disulfide bonds. N-terminal sequence analyses of tuatara α₂-M revealed a distinct similarity to α-macroglobulins of other vertebrates and that at least two types of α₂-M subunits are present in plasma of tuatara.     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Electron transfer by b- and c-type cytochromes in relation to the origin of the ‘slow’ electric potential component

Cytochrome redox changes and electric potential generation are kinetically compared during cyclic electron transfer in Photosystem-I-enriched and Photosystem-II-depleted subchloroplast vesicles (i.e., stroma lamellae membrane vesicles) supplemented with ferredoxin using a suitable electron donating system. In response to a single-turnover flash, the sequence of events is: (1) fast reduction of cytochrome b-563 (t_0.5 ≈ 0.5 ms) (2) oxidation of cytochrome c-554 (t_0.5 ≈ 2 ms), (3) slower reduction of cytochrome b-563 (t_0.5 ≈ 4 ms), (4) generation of the ‘slow’ electric potential component (t_0.5 ≈ 15–20 ms), (5) re-reduction of cytochrome c-554 (t_0.5 ≈ 30 ms) and (6) reoxidation of cytochrome b-563t_0.5 ≈ 90 ms). Per flash two cytochrome b-563 species turn over for one cytochrome c-554. These b-563 cytochromes are reduced with different kinetics via different pathways. The fast reductive pathway proceeds probably via ferredoxin, is insensitive to DNP-INT, DBMIB and HQNO and is independent on the dark redox state of the electron transfer chain. In contrast, the slow reductive pathway is sensitive to DNP-INT and DBMIB, is strongly delayed at suboptimal redox poising (i.e., low $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ ratio) and is possibly coupled to the reduction of cytochrome c-554. Each reductive pathway seems obligatory for the generation of about 50% of the slow electric potential component. Also cytochrome c-559_LP (LP, low potential) is involved in Photosystem-I-associated cyclic electron flow, but its flash-induced turnover is only observed at low preestablished electron pressure on the electron-transfer chain. Data suggest that cyclic electron flow around Photosystem I only proceeds if cytochrome b-559_LP is in the reduced state before the flash, and a tentative model is presented for electron transfer through the cyclic system.     

A 250-kilodalton cellular protein is induced by progestins in two human breast cancer cell lines MCF7 and T47D

We have studied the effect of R5020, a synthetic progestin, on the biosynthesis of cellular proteins extracted from the MCF7 and T47D human breast cancer cells, using gel electrophoresis. R5020 stimulates the synthesis, as measured after [35S]-methionine labelling, and the accumulation, as shown by silver staining, of a protein of molecular weight approximately equal to 250,000. The increase of the labelled 250-kilodalton protein was rapid (3 hours) and after 3 days this protein represented approximately equal to 6% of the total cellular proteins (approximately equal to 1 microgram/150,000 cells). The induction of the 250-kilodalton protein was obtained by physiologically active concentrations of several progestins and high concentrations of 5 alpha-dihydrotestosterone but not by estradiol or dexamethasone. It was inhibited by R486 , a progestin antagonist, but not by flutamide, an androgen antagonist. These results indicate a mediation by the progesterone receptor. The 250-kilodalton protein appears to be an excellent probe to study in cell culture the mechanism of action of progestin on human cells.     

Purification and properties of the H2-oxidizing (uptake) hydrogenase of the N2-fixing anaerobe Clostridium pasteurianum W5

Clostridium pasteurianum has two distinct hydrogenases, the bidirectional hydrogenase and the H₂-oxidizing (uptake) hydrogenase. The H₂-oxidizing hydrogenase has been purified (up to 970-fold) to a specific activity of 17,600 μmol H₂ oxidized/min·mg protein (5 mM methylene blue) or 3.5 μmol H₂ produced/min·mg protein (1 mM methyl viologen). The uptake hydrogenase has a M_r of 53,000 (one polypeptide chain). Depending upon how protein was measured, the Fe and S⁼ contents (gatom/mol) were 4.7 and 5.2 (by the dye-binding assay) or 7.2 and 8.0 (by the Lowry method). Both reduced and oxidized forms of the enzyme gave electron paramagnetic resonance signals. The activation energy for H₂-production and H₂-oxidation by the uptake hydrogenase was 59.1 and 31.2 kJ/mol, respectively. In the exponential phase of growth, the ratio of uptake hydrogenase/bidirectional hydrogenase in NH₃-grown cells was much lower than that in N₂-fixing cells.     

Relationships between the effects of redox potential, alpha-thenoyltrifluoroacetone and malonate on O(2) and H2O2 generation by submitochondrial particles in the presence of succinate and antimycin

A successful approach has been developed for the sequencing of apolipoprotein B based upon the procedure of Cleveland et al. [(1977) J. Biol. Chem. 252, 1102-1106] involving limited proteolysis in the presence of sodium dodecyl sulfate. Staphylococcus aureus protease was employed to produce large peptides which were isolated in relatively pure form by preparative gel electrophoresis. Two peptides were partially sequenced using spinning-cup microsequencing techniques. The sequences are: Peptide R2-5, -Ala-Leu-Val-Gly-Ile-Asn- Gly-Glu-Ala-Asn-Leu-Asp-Phe-Leu-Asn-Ile-Pro-Leu-Arg-Ile-Pro-Pro- Met-Arg-(Arg)-; Peptide R3-1, -Leu-Val-Ala-Lys-Pro-Ser-Val-Ser-Val-Glu- Phe-Val-Thr-Asn-Met-Gly-Ile-Ile-Pro-Lys-Phe-Ala-Arg-. Several stretches of residues suitable for the construction of oligonucleotide probes have been identified.     

Systemic effects of chronically administered methyl prednisolonate and methyl 17-deoxyprednisolonate

The systemic activities of methyl prednisolonate and methyl 17-deoxyprednisolonate (1) were studied in rats. Methyl 17-deoxyprednisolonate produced significant changes in the amount of sodium ion (decreased) and potassium ion (increased) in urine; however, methyl prednisolonate had no effect on electrolyte balance. Both methyl prednisolonate and methyl 17-deoxyprednisolonate had no effect on liver glycogen content, plasma corticosterone level and relative adrenal weight. In contrast, the parent compound prednisolone caused a significant decrease in liver glycogen content, plasma corticosterone level and relative adrenal weight.