Combinatorial regulation of the balance between dynein microtubule end accumulation and initiation of directed motility

Cytoplasmic dynein is involved in a multitude of essential cellular functions. Dynein's activity is controlled by the combinatorial action of several regulatory proteins. The molecular mechanism of this regulation is still poorly understood. Using purified proteins, we reconstitute the regulation of the human dynein complex by three prominent regulators on dynamic microtubules in the presence of end binding proteins (EBs). We find that dynein can be in biochemically and functionally distinct pools: either tracking dynamic microtubule plus‐ends in an EB‐dependent manner or moving processively towards minus ends in an adaptor protein‐dependent manner. Whereas both dynein pools share the dynactin complex, they have opposite preferences for binding other regulators, either the adaptor protein Bicaudal‐D2 (BicD2) or the multifunctional regulator Lissencephaly‐1 (Lis1). BicD2 and Lis1 together control the overall efficiency of motility initiation. Remarkably, dynactin can bias motility initiation locally from microtubule plus ends by autonomous plus‐end recognition. This bias is further enhanced by EBs and Lis1. Our study provides insight into the mechanism of dynein regulation by dissecting the distinct functional contributions of the individual members of a dynein regulatory network.     

植物数量性状遗传体系检测中回交或自交家系重复试验数据的分析方法

为提高主基因 多基因混合遗传分析的精度 ,降低试验误差 ,采用重复内分组随机区组设计 ,对低遗传力性状的B1∶2 和B2∶2 或F2∶3 家系平均数资料进行遗传分析。通过AIC准则和适合性检验比较无主基因 (A - 0 )、1对主基因 (A)、2对主基因 (B)、多基因 (C)、1对主基因 多基因 (D)和 2对主基因 多基因 (E)模型以鉴定其遗传模式。采用IECM算法估计混合模型参数。通过油菜HSTC14×宁油 7号初花期F2∶3 家系平均数资料阐明该方法。     

Radioisotopic assay of femtomole quantities of total adenine nucleotides,ATP plus ADP,and AMP

AMP is converted to ATP by incubating overnight with pyruvate kinase, phosphoenolpyruvate and adenylate kinase in th prensence of endogenous ATP (ADP) as primer. In a subsequent incubation in the presence of pyruvate kinase, phosphoenolpyruvate, radioactive glucose and hexokinase. ATP and ADP are estimated together by coupling their recycling to the formation of glucose 6-phosphate. The latter is separated by precipitation using 76% (v/v) acetone for radioactivity measurement in the same Eppendorf tube. The sensitivity of these simple procedures matches or exceeds those of luciferase methods of nucleotide determination.     

Metallothionein-I induction by stress in specific brain areas

The distribution of metallothionein-I (MT) in several areas of the brain and its induction by immobilization stress has been studied in the rat. MT content was highest in hippocampus and midbrain and lowest in frontal cortex and pons plus medulla oblongata. Immobilization stress for 18 hours (which was accompanied by food and water deprivation) significantly increased MT levels in the frontal cortex, pons plus medulla oblongata and hypothalamus, but not in midbrain and hippocampus. The effect of stress on MT levels was specific as food and water deprivation along had no significant effect on MT levels in any of the brain areas studied. The effect of stress on MT levels was independent of changes in cytosolic Zn content; this was generally unaffected by stress or food and water deprivation but decreased in pons plus medulla oblongata from stressed rats. The results suggest that MT is induced more significantly in the brain areas that are usually involved in the response of animals to stress.     

Chitin synthesis in larval and pupal epidermis of the Indian meal moth,Plodia interpunctella (Hübner), and the greater wax moth,Galleria mellonella (L.)

The level of chitin synthesis was determined in whole last-instar larvae and in pupae of Plodia interpunctella, and in epidermal tissue from similar stages of Galleria mellonella. The incorporation of radiolabelled N-acetyl-d-glucosamine into chitin was used to measure synthesis. Chitin production was similar in both species with peaks of synthesis occurring at the beginning of the last larval instar, in prepupae, in white pupae and prior to adult emergence. P. interpunctella also exhibited an additional small increase at mid-instar. Exposure of larval epidermis of P. interpunctella to 20-hydroxyecdysone in vitro stimulated chitin synthesis, but only after a 24 hr lag period subsequent to exposure to the hormone. This hormonal stimulation of chitin synthesis was inhibited by actinomycin-D and cycloheximide which suggested that 20-hydroxyecdysone-stimulated production of chitin depended on synthesis of RNA and protein. Comparison of the synthesis of chitin in epidermis of G. mellonella with previously published ecdysone titres, indicated that chitin production in vivo is preceded by an elevated ecdysone titre.     

Evidence for a reactive sulfhydryl in the DNA binding domain of the 1,25-dihydroxyvitamin D3 receptor

Evidence is presented here that organomercurial binding to a reactive sulfhydryl group is capable of altering the DNA-binding characteristics of the 1,25-dihydroxyvitamin D receptor (D-receptor). Accordingly, hormone-free receptor (R_o) binding to DNA-cellulose is inhibited in a concentration-dependent fashion with both HgCl₂ and p-chloromercuribenzene sulfonate (pCMBS) with complete inhibition evident at 1.0 mM. Further, low concentrations (0.5 mM) of mercurials are also capable of dissociating preformed DNA-receptor complexes, a process reversible with excess thiol reagent such as monothioglycerol. These findings are in contrast to alkylating reagents such as iodoacetamide, which is capable of only partially inhibiting the formation of the receptor-DNA duplex (37% at 25 mM). Once created, however, the duplex is completely insensitive to dissociation (even at 25 mM). These results imply that in addition to the association of a cysteine(s) moiety in or near the sterol binding site, modification of a similarly reactive group(s) can also alter the D-receptor's DNA-binding domain.     

Structure of indole-3-acetic acid myoinositol esters and pentamethyl-myoinositols

GC-MS properties of three isomeric esters of indole-3-acetic acid and myoinositol, three esters of indole-3-acectic acid and myoinositol arabinoside and three esters of indole-3-acetic acid and myoinositol galactoside are presented. MS fragmentation patterns for the four possible pentamethyl myoinositols are also shown. These data indicated that the arabinose, and galactose of the glycosides were in the pyranose form and that C-1 of the sugar was linked to the 5 hydroxyl of myoinositol. Homologies in fragmentation patterns for the esters and the glycoside esters, together with knowledge of the properties of 2-O-indole-3-acetyl-myoinositol, permitted identification of one of the arabinosides as 5-O-l-arabinopyranosyl-2-O-indole-3-acetyl-myoinositol and one of the galactosides as 5-O-d- galactopyranosyl-2-O-indole-3-acetyl-myoinositol. The remaining two GLC peaks observed for the arabinoside were then, most likely, the two mixtures of diastereoisomers 1 d- and 1 l-5-O-l-arabinopryranosyl-1-O-indole-3-acetyl myoinositol and 1 d- and 1 l-5-O-l-arabinopyranosyl-4-O-indole-3-acetyl-myoinositol. The remaining two GLC peaks observed for the galactoside would then be the 1 d and 1 l-5-O-d-galactopyranosyl-1-O-indole-3-acetyl-myoinositol and 1 d- and 1 l-5-O-d- galactopyranosyl-4-O-indoleacetyl-myoinositol.     

The influence of serum components on the growth and mutation of Chinese hamster cells in medium containing 8-azaguanine

Low concentrations (?20 μg/ml) of 8-azaguanine are 1000 fold more toxic to V79 Chinese hamster cells in medium containing 10% dialyzed fetal calf serum than in medium containing 10% undialyzed serum. Serum enzyme activity that converts AG to nontoxic 8-azaxanthine degrades AG at the same rate, whether or not the serum is dialyzed. However, cytotoxicity results similar to those obtained with US were produced in medium containing DS and 2.5 μg of hypoxanthine (HX)/ml (DSH). Therefore, serum HX is considered to be responsible for the relatively low cytotoxicity of AG in medium containing US. Colonies that arose in medium containing AG were isolated and characterized. Those that remained resistant to AG (40 μg/ml) and sensitive to aminopterin in the presence of HX and thymidine (HAT) were considered mutants; nonmutants were sensitive to AG and resistant to HAT. Colonies isolated from medium containing DSH of US and low concentrations of AG were not mutants, but those from medium containing high concentrations (? μg/l) of AG were mutants. Spontaneous and N-methyl-N′-nitrosoguanidine induced mutants were detectable in medium containing DSH without replating the cells prior to adding AG (?30 μg/ml), but in order to detect MNNG induced mutations in medium containing DS replating was essential. In DS, the mutation frequency increased as an exponential function of the toxicity of MNNG, but remained two orders of magnitude lower than the induced mutation frequencies that occurred in DSH, HX, in DSH or US, produced profound effects, other than interference with AG toxicity, that distort the results of mutagenesis assays. To study mutation using AG resistance as the endpoint, it is essential to use dialyzed serum.     

A comparison of cytotoxicity, ouabain-resistant mutation, sister-chromatid exchanges, and nascent DNA synthesis in Chinese hamster cells treated with dihydrodiol epoxide derivatives of benzo[a]pyrene

Chinese hamster V79 cells were treated with either (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P-diol epoxide I) or (+/-)-7 beta,8 alpha-dihydroxy-9 beta,10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P-diol epoxide II) and the nascent DNA was labeled with [Me-3H]thymidine. The cells were harvested for determination of cytotoxicity, sister-chromatid exchanges (SCE), ouabain-resistant (Or) mutations and the size of newly synthesized daughter-strand DNA. Both isomers caused dose-dependent decreases in survival of cells and in the size of nascent DNA. Increases in the frequencies of SCE and of Or mutation were found in cells treated with either isomer. However, B[a]P-diol epoxide I caused 10--20-fold more Or mutations and 50-100% more SCE than did B[a]P-diol epoxide II at equal molar dose levels. In contrast to the marked difference in the frequencies of both SCE and Or mutations caused by both compounds, the isomers induced similar reductions in the size of the nascent DNA at equal dose levels. In comparing the molecular and biological effects of the two isomers the reduction in the size of nascent DNA was more closely related to cytotoxicity than to the induction of SCE or Or mutations.