Antitumor agents-CLXXV. Anti-tubulin action of (+)-thiocolchicine prepared by partial synthesis

(+)-Thiocolchicine (2b) was prepared from (±)-colchicine (1) in a five-step reaction sequence that included chromatographic separation of appropriate camphanylated diastereomers. Acid hydrolysis of the (+)-diastereomer, followed by acetylation, yielded the desired product 2b. (+)-Thiocolchicine has 15-fold lower inhibitory activity against tubulin polymerization than (−)-thiocolchicine, and is 29-fold less potent for inhibiting growth of human Burkitt lymphoma cells. The enantiomer 2a, prepared from the (−)-camphanylated diastereomer, had potent activity in all assays comparable to that of (−)-thiocolchicine prepared by other methods. These results support the hypothesis that the proper configuration of colchicine-related compounds is an important requirement for their anti-tubulin action.     

不同光学异构体组成的氯氰菊酯对敏感及抗性家蝇的神经电生理学研究

采用敏感家蝇(Musca domestica vicina L.)及由5种不同光学异构体组成的氯氰菊酯选育的抗性家蝇中胸足离体标本,观测五种药剂对足感觉神经纤维冲动发放的影响。各种异构体组成的氯氰菊酯均可引起感觉神经纤维发放的增加,然后逐渐降低,直至完全阻断。以阻断时间和加药剂量为参数,求出神经敏感度。结果表明：五种药剂作用于敏感家蝇的神经敏感度与室内生物测定的LD₅₀值(μg/头)无相关性,而与供试药剂中反α体与顺α体的比例有关,反α体所占比例越多的药物,神经敏感度越高。抗性家蝇的神经敏感度与敏感 家蝇相比大幅度下降,可以认为神经敏感度降低是家蝇对氯氰菊酯产生抗性的主要机制。抗性家蝇中,氯氰菊酯品系(RC₁)的抗生水平最高,但它的神经敏感度较其它抗性品系也高,由此推测,RC₁,的抗性机制与其它晶系有所不同。     

Covalently cyclized agonist and antagonist analogues of bombesin and related peptides.

During a search for possible cyclization points in shortened, potent bombesin agonists and antagonists, it was found that the joining of amino acid residues in positions 6 and 14 by various means resulted in retention of significant binding affinity for rat pancreatic acini and murine Swiss 3T3 cells. In one series of analogues, Cys residues in these positions were used for bridging via a disulfide bond. (D)-C-Q-W-A-V-G-H-L-C-NH2 retained significant binding affinity for rat pancreatic acini cells and was a full amylase releasing agonist (EC50 187 nM). Potency was markedly increased by substituting D-Ala for Gly (EC50 67 nM compared to 10 nM for its linear counterpart) and was decreased by substituting L-Cys for D-Cys in this analogue (EC50 214 nM), thus strongly suggesting stabilization of peptide folding by the D residues. Elimination of the COOH-terminal amino acid produces competitive antagonists in the linear analogues; however, (D)-C-Q-W-A-V-G-H-C-NH2 was devoid of activity. Likewise, cyclization to position 13 with the 14 amino acids intact to give (D)-C-Q-W-A-V-G-H-C-L-NH2 resulted in an almost inactive peptide. On the other hand, as in the linear series, the reduced peptide bond analogue, (D)-C-Q-W-A-V-(D)-A-H-L-psi (CH2NH)-C-NH2, was a receptor antagonist (IC50 5.7 mM), albeit much weaker than the corresponding linear analogues, but with no residual agonist activity. Direct head-to-tail cyclization was also tried. Both cyclo[(D)-F-Q-W-A-V-G-H-L-L] (EC50 346 nM) and the shorter cyclo [Q-W-A-V-G-H-L-L] (EC50 1236 nM) were full agonists. Elimination of the COOH-terminal residue in cyclo[(D)-p-Cl-F-Q-W-A-V-(D)-A-H-L] produced an agonist (EC50 716 nM) rather than an antagonist. These results provide support for the proposal that both bombesin agonists and antagonists adopt a folded conformation at their receptor(s). Furthermore, the retention of appreciable potencies using several cyclization strategies and chain lengths suggests that further optimization of these structures both in terms of potency and ring size is possible. Since these peptides have increased conformational restriction, they should begin to serve as useful substrates for NMR and molecular modeling studies aimed at comparing the obviously subtle differences between agonist and antagonist structures.     

Induction of the second exopolysaccharide (EPSb) in Rhizobium meliloti SU47 by low phosphate concentrations.

In previous work, Rhizobium meliloti SU47 produced its alternative exopolysaccharide (EPSb [also called EPS II]) only in strains that were genetically altered to activate EPSb synthesis. Here we report that EPSb synthesis is not entirely cryptic but occurred under conditions of limiting phosphate. This was shown in several different exo mutants that are blocked in the synthesis of the normal exopolysaccharide, succinoglycan. In addition, EPSb biosynthetic gene expression was markedly increased by limiting phosphate. An apparent regulatory mutant that does not express alkaline phosphatase activity was unable to produce EPSb under these conditions. A mucR mutant that was previously shown to produce EPSb instead of the normal exopolysaccharide, succinoglycan, was not sensitive to phosphate inhibition of EPSb synthesis. No evidence was found to indicate that exoX, which affects succinoglycan synthesis, had any influence on EPSb synthesis. In contrast to limiting phosphate, limiting nitrogen or sulfur did not stimulate EPSb synthesis as it does succinoglycan.     

Reduced susceptibility to HIV-1 infection of ethyl-methanesulfonate-treated CEM subclones correlates with a blockade in their protein kinase C signaling pathway.

We have described the isolation of chemically induced CEM subclones that express CD4 receptors and bind soluble gp120, yet show a markedly reduced susceptibility to infection with HIV-1. Two subclones were found to have an abnormal response to the protein kinase C (PKC) activator PMA. PMA treatment induced CD3 and CD25 (IL-2R) receptors on the parental line and on other ethyl-methanesulfonate-derived subclones, but not on these two mutants. Direct assays of PKC activity were conducted. Total cellular PKC enzymatic activity was found to be normal in these subclones. PMA-induced CD4 down-modulation occurred normally. In addition, activation of c-raf kinase was normal. Since HIV-1 long terminal repeat contains two functional nuclear factor kB (NF-kB) regulatory elements, we studied the ability of PMA to induce NF-kB binding activity by different assays. Chloramphenicol acetyl transferase (CAT) assays using the HIV-1 (-139)long terminal repeat-CAT construct showed no PMA induction of CAT activity in these subclones (unlike the parental line and other subclones). Okadaic acid, an inhibitor of phosphatases 1 and 2A, did not overcome the defect in these subclones. Gel retardation assays, using a 32P-probe containing the HIV-1 NF-kB probe and nuclear extracts from PMA-treated cells, showed significantly reduced induction of nuclear NF-kB binding proteins in these two subclones compared with wild type CEM and a control subclone. Deoxycholate treatment of cytoplasmic extracts from these subclones released much reduced NF-kB binding proteins from their cytoplasmic pools. Thus, reduced levels of PKC-induced nuclear NF-kB activity in two T cell subclones did not affect their normal cell growth, but correlated with a pronounced reduction in their susceptibility to HIV-1 infection.     

Pulmonary vascular reactivity and hemodynamic changes in elastase-induced emphysema in hamsters.

Changes in pulmonary hemodynamics and vascular reactivity in emphysematous hamsters were studied in an isolated lung preparation perfused at constant flow with blood and 3% dextran. Hamsters were treated with intratracheal porcine pancreatic elastase at 70 days of age, and experimental studies were conducted at 1, 3, and 8 mo after treatment. Baseline pulmonary arterial pressure in elastase-treated lungs was increased compared with saline-treated control lungs 1 mo after treatment, but this increase did not progress at 3 and 8 mo. Increases in pulmonary arterial pressure in elastase-treated lungs were temporally correlated with the morphological development of emphysema and right ventricular hypertrophy; both of these were evident at 1 mo after treatment and showed little change thereafter. Pressor responses to hypoxia and angiotensin II were not different between elastase-treated and control lungs at 1 and 3 mo. At 8 mo, however, pressor responses in emphysematous lungs to 0% O2 (but not to angiotensin II) were significantly increased. This was the result of a lack of the normal age-related fall in the hypoxic pressor response. Our results suggest that the right ventricular hypertrophy found in these emphysematous animals results from a chronically increased pulmonary vascular resistance. Furthermore, increases in pulmonary vascular resistance in the early development of emphysema are likely a result of the loss of vascular beds and supporting connective tissue.     

Functional and evolutionary relatedness of genes for exopolysaccharide synthesis in Rhizobium meliloti and Rhizobium sp. strain NGR234.

Rhizobium meliloti SU47 and Rhizobium sp. strain NGR234 produce distinct exopolysaccharides that have some similarities in structure. R. meliloti has a narrow host range, whereas Rhizobium strain NGR234 has a very broad host range. In cross-species complementation and hybridization experiments, we found that several of the genes required for the production of the two polysaccharides were functionally interchangeable and similar in evolutionary origin. NGR234 exoC and exoY corresponded to R. meliloti exoB and exoF, respectively. NGR234 exoD was found to be an operon that included genes equivalent to exoM, exoA, and exoL in R. meliloti. Complementation of R. meliloti exoP, -N, and -G by NGR234 R'3222 indicated that additional equivalent genes remain to be found on the R-prime. We were not able to complement NGR234 exoB with R. meliloti DNA. In addition to functional and evolutionary equivalence of individual genes, the general organization of the exo regions was similar between the two species. It is likely that the same ancestral genes were used in the evolution of both exopolysaccharide biosynthetic pathways and probably of pathways in other species as well.     

Two genes that regulate exopolysaccharide production in Rhizobium meliloti.

We describe a new Rhizobium meliloti gene, exoX, that regulates the synthesis of the exopolysaccharide, succinoglycan, exoX resembled the psi gene of R. leguminosarum bv. phaseoli and the exoX gene of Rhizobium sp. strain NGR234 in its ability to inhibit exopolysaccharide synthesis when present in multiple copies, exoX did not appear to regulate the expression of exoP. The effect of exoX was counterbalanced by another R. meliloti gene, exoF. exoF is equivalent to Rhizobium sp. strain NGR234 exoY and resembles R. leguminosarum bv. phaseoli pss2 in its mutant phenotype and in portions of its deduced amino acid sequence. The effect of exoF on the succinoglycan-inhibiting activity of exoX depended on the relative copy numbers of the two genes. exoX-lacZ fusions manifested threefold-higher beta-galactosidase activities in exoF backgrounds than in the wild-type background. exoX mutants produced increased levels of succinoglycan. However, the exoF gene was required for succinoglycan synthesis even in an exoX mutant background. exoF did not affect the expression of exoP. Strains containing multicopy exoX formed non-nitrogen-fixing nodules on alfalfa that resembled nodules formed by exo mutants defective in succinoglycan synthesis. exoX mutants formed nitrogen-fixing nodules, indicating that, if the inhibition of succinoglycan synthesis within the nodule is necessary for nitrogen fixation, then exoX is not required for this inhibition. We present indirect evidence that succinoglycan synthesis within the nodule is not necessary for bacteroid function.     

荒漠草原盐沼湿地苦豆子土壤细菌群落构建机制及其影响因素

【背景】荒漠草原盐沼湿地是陆地生态系统的重要组成部分,土壤水分和盐分变化是影响该生态系统土壤细菌群落构建的重要因素。【目的】土壤细菌群落构建是由确定性和随机性主导的连续生态过程,阐明荒漠草原盐沼湿地土壤细菌群落的构建机制对于加深微生物作为关键生态系统因子重要性的理解具有积极意义。【方法】以宁夏中部典型荒漠草原盐沼苦水湖湿地为研究对象,对近湖边（near the lake,NL）和远离湖边（far from the lake,FL）苦豆子群落土壤理化特性进行测定并结合土壤细菌高通量测序分析。【结果】NL和FL样地具有明显的水盐梯度变化,NL样地土壤pH、含水量和电导率均显著高于FL样地;变形菌门、放线菌门、厚壁菌门、拟杆菌门和黏菌门是研究区域土壤细菌群落的优势菌门,变形菌门相对丰度随水盐梯度上升而升高,放线菌门和厚壁菌门相对丰度则随之下降,门下成员大多与水盐变化具有明显的相关性;此外,FL样地土壤细菌网络则具有稳定的网络关系;随着NL样地向FL样地的延伸,土壤细菌群落由随机过程主导,并且受pH、电导率和环境变量的影响。【结论】荒漠草原盐沼湿地水分和盐分的变化改变了土壤细菌群落结构;土壤细菌群落通过生态位占据等策略提高逆境下的生存能力;细菌群落构建是随机过程和确定性过程组成的连续统一体,同样受环境变化的影响。本结果揭示了荒漠草原盐沼湿地细菌群落结构和相互关系对环境变化的响应特征,同时阐明了该区土壤细菌群落的构建机制及影响因素,也为相关科学研究提供了一定理论参考。