Transient kinetics of the interaction of actin with myosin subfragment-1 in the absence of nucleotide.

The kinetics of the association of actin with myosin subfragment-1 (S1) has been studied by using S1 labeled at the sulfhydryl group SH1 with 5-(iodoacetamido)fluorescein (S1-AF). Upon rapid mixing in a stopped-flow apparatus, the fluorescence intensity of the fluorescein moiety increased by 50%, followed by a slower increase that was well resolved. This slow phase of the fluorescence change could not be fitted to either a monoexponential or a biexponential function, but it could be fitted to a sum of three exponential terms yielding three observed first-order rate constants (lambda i). The dissociation of acto.-(S1-AF) was studied by displacement of S1-AF from the complex with native S1. The dissociation kinetics was characterized by a single rate constant (approximately 0.012 s-1 at 20 degrees C), and this constant was independent of S1 concentration. Together with previous equilibrium data that were obtained under identified conditions for formation of acto-subfragment-1 (Lin, S.-H., and H. C. Cheung. 1991. Biochemistry. 30:4317-4323), a six-state two-pathway model is proposed as a minimum kinetic scheme for formation of rigor acto.S1. In this model, unbound subfragment-1 exists in two conformational states (S1' and S1) which are in equilibrium with each other, one corresponding to the previously established low-temperature state and the other to the high-temperature state. Each subfragment-1 state can interact with actin to form a collision complex, followed by two isomerizations to form two acto-subfragment-1 states (A.S1' and A.S1). Both isomerizations were visible in stopped-flow experiments. Two special cases of the model were considered: 1) a rapid pre-equilibration of the initial collision complex with actin and S1, and 2) trace accumulation of the collision complex. The first case required that the three combinations of the three observed rate constants be independent of actin concentration. The data were incompatible with this approximation. The other special case required that the sum of the lambda i vary linearly with actin concentration and the other two combinations of lambda i vary with actin concentration in a quadratic fashion. The present data were in agreement with the second case. At 20 degrees C and in 60 mM KCl, 2 mM MgCl2, 30 mM 2-([-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino)ethanesulfonic acid, and pH 7.5, the biomolecular association rate constants for the interaction of actin with S1' and S1 were 8.58 x 10(5) and 1.11 x 10(6) M-1 s-1, respectively.     

Isolation and identification of a novel tandemly repeated DNA sequence in the centromeric region of human chromosome 8

EcoRI subclones, designated as 50E1 and 50E4, were independently obtained from a cosmid clone previously mapped to the centromeric region of human chromosome 8. Southern blot hybridization analyses suggested that both subclones contain repetitive DNA sequences different from the chromosome 8 specific alphoid DNA. DNA sequence analysis of the 704 bp insert of 50E1 and the 1, 962 bp insert of 50E4 revealed that both inserts contained tandemly repeated units of 220 bp. Fluorescence in situ hybridization studies confirmed these two subclones to be specifically located on the centromeric region of chromosome 8. A 220 bp consensus sequence, derived from nine monomeric repeats, showed no significant homology to alphoid consensus sequences or to other currently known human centromeric DNA sequence. Furthermore, no significant homology was found with any other DNA sequence deposited in the EMBL or GenBank databases, indicating that this chromosome 8 specific repetitive DNA sequence is novel. From slot blot experiments it was estimated that 0.013% of the human genome comprises 1,750 of these monomeric repeats, residing on the centromeric region of chromosome 8 in tandem array(s).     

Lysophosphatidylcholine disrupts the acrosome of tammar wallaby (macropus eugenii) spermatozoa

The acrosomal status of wallaby spermatozoa was evaluated by light and electron microscopy after incubation in 1–100 μM lysophosphatidylcholine (LPC) for up to 120 min. Treatment with 1 and 10 μM LPC for 120 min did not lead to acrosomal loss, or detectable alteration to the acrosome, as detected by Bryan's staining and light microscopy. Incubation with 25 μM LPC had little effect on acrosomal loss, however statistically significant changes (P < 0.05) in the acrosomal matrix (altered) were detected after 10-min incubation by light microscopy. Around 50% of acrosomes were altered after 20-min incubation in 50 μM LPC (P < 0.001), and 40% of spermatozoa had lost their acrosome after 60-min incubation (P < 0.001). Treatment with 75 and 100 μM LPC led to rapid acrosomal loss from around 50% of spermatozoa within 10 min (P < 0.001), and by 60 min acrosomal loss was 70–80%. LPC, like the diacylglycerol DiC₈ (1,2-di-octanoyl-sn-glycerol), is thus an effective agent to induce loss of the relatively stable wallaby sperm acrosome, and it also induces changes within the acrosomal matrix. Ultrastructure of the LPC-treated spermatozoa revealed that the plasma membrane and the acrosomal membranes were disrupted in a manner similar to that seen after detergent treatment (Triton X-100). There was no evidence of point fusion between the plasma membrane overlying the acrosome and the outer acrosomal membrane. The plasma membrane was the first structure to disappear from the spermatozoa. The acrosomal membranes and matrix showed increasing disruption with time and LPC concentration. Wallaby spermatozoa incubated with LPC at concentrations that induced significant acrosomal loss also underwent a rapid decline in motility that suggested that acrosomal loss may be due to cell damage, rather than a physiological AR. This study concluded that LPC-induced acrosomal loss from tammar wallaby spermatozoa is due to its action as a natural detergent and not as a phosphoinositide pathway intermediate. The study further demonstrates the unusual stability of the marsupial acrosomal membranes. © 1993 Wiley-Liss, Inc.     

利用酵母菌生产有机酸的研究进展

有机酸是含有一种或多种低分子量酸性基团(如羧基、磺酸基)的可生物合成的有机化合物,广泛应用于食品、农业、医药、生物基材料工业等领域。酵母菌具有生物安全、抗逆性强、底物谱广泛、方便遗传改造,以及大规模培养技术成熟等独特优点,因此利用酵母菌生产有机酸的研究日益受到国内外学者的关注。目前利用酵母生产有机酸还存在浓度低、副产物多,以及发酵效率低等缺陷。随着酵母菌代谢工程和合成生物学技术的发展,利用酵母菌生产有机酸取得了快速进展。本文总结了利用酵母合成11种有机酸的研究,包括内源和异源合成的大宗羧酸和高价值有机酸,并对该领域的未来研究方向进行了展望。     

Rapid microbial growth in a pH auxostat

Summary Feeding nutrient to meet demand dilutes slow-growing organisms from continuous culture and greatly favors rapid growth. Doubling times of roughly 10 minutes have been verified in a pH auxostat by viable cell counts and by direct counting with a Petroff-Hausser chamber. Effects of wall attachment were negligible because a fresh reactor was substituted frequently before wall growth could become established.     

Design considerations for two-stage enrichment clinical trials

When there is a predictive biomarker, enrichment can focus the clinical trial on a benefiting subpopulation. We describe a two-stage enrichment design, in which the first stage is designed to efficiently estimate a threshold and the second stage is a “phase III-like” trial on the enriched population. The goal of this paper is to explore design issues: sample size in Stages 1 and 2, and re-estimation of the Stage 2 sample size following Stage 1. By treating these as separate trials, we can gain insight into how the predictive nature of the biomarker specifically impacts the sample size. We also show that failure to adequately estimate the threshold can have disastrous consequences in the second stage. While any bivariate model could be used, we assume a continuous outcome and continuous biomarker, described by a bivariate normal model. The correlation coefficient between the outcome and biomarker is the key to understanding the behavior of the design, both for predictive and prognostic biomarkers. Through a series of simulations we illustrate the impact of model misspecification, consequences of poor threshold estimation, and requisite sample sizes that depend on the predictive nature of the biomarker. Such insight should be helpful in understanding and designing enrichment trials.     

Chromosomal localization of uroplakin genes of cattle and mice

The asymmetric unit membrane (AUM) of the apical surface of mammalian urinary bladder epithelium contains several major integral membrane proteins, including uroplakins IA and IB (both 27 kDa), II (15 kDa), and III (47 kDa). These proteins are synthesized only in terminally differentiated bladder epithelial cells. They are encoded by separate genes and, except for uroplakins IA and IB, appear to be unrelated in their amino acid sequences. The genes encoding these uroplakins were mapped to chromosomes of cattle through their segregation in a panel of bovine x rodent somatic cell hybrids. Genes for uroplakins IA, IB, and II were mapped to bovine (BTA) Chromosomes (Chrs) 18 (UPK1A), 1 (UPK1B), and 15 (UPK2), respectively. Two bovine genomic DNA sequences reactive with a uroplakin III cDNA probe were identified and mapped to BTA 6 (UPK3A) and 5 (UPK3B). We have also mapped genes for uroplakins 1A and II in mice, to the proximal regions of mouse Chr 7 (Upk1a) and 9 (Upk2), respectively, by analyzing the inheritance of restriction fragment length variants in recombinant inbred mouse strains. These assignments are consistent with linkage relationships known to be conserved between cattle and mice. The mouse genes for uroplakins IB and III were not mapped because the mouse genomic DNA fragments reactive with each probe were invariant among the inbred strains tested. Although the stoichiometry of AUM proteins is nearly constant, the fact that the uroplakin genes are unlinked indicates that their expression must be independently regulated. Our results also suggest likely positions for two human uroplakin genes and should facilitate further analysis of their possible involvement in disease.     

PDGF-AA activates AKT and ERK signaling for testicular interstitial Leydig cell growth via primary cilia

Testes control the development of male reproductive system. The testicular interstitial Leydig cells (Leydig cells) synthesize testosterone for promoting spermatogenesis and secondary sexual characteristics. Type A platelet-derived growth factor (PDGF-AA) is one of the most important growth factors in regulating Leydig cell growth and function. Knockout of PDGF-AA or its congenital receptor PDGFR-α leads to poor testicular development caused by reducing Leydig cell numbers, supporting PDGF-AA/PDGFR-α signaling regulates Leydig cell development. Primary cilium is a cellular antenna that functions as an integrative platform to transduce extracellular signaling for proper development and differentiation. Several receptors including PDGFR-α are observed on primary cilia for initiating signaling cascades in distinct cell types. Here we showed that PDGF-AA/PDGFR-α signaling promoted Leydig cells growth, migration, and invasion via primary cilia. Upon PDGF-AA treatment, AKT and ERK signaling were activated to regulate these cellular events. Interestingly, active AKT and ERK were detected around the base of primary cilia. Depletion of ciliary genes (IFT88 and CEP164) alleviated PDGF-AA-activated AKT and ERK, thus reducing Leydig cell growth, migration, and invasion. Thus, our study not only reveals the function of PDGF-AA/PDGFR-α signaling in maintaining testicular physiology but also uncovers the role of primary cilium and downstream signaling in regulating Leydig cell development.     

褪黑素调节烟草丁香假单胞菌抗性的功能研究

为分析褪黑素(N-乙酰-5-甲氧基色胺)在植物先天免疫中的功能及调控机理,研究以病原菌丁香假单胞杆菌(Pseudomonas syringae pv.tomato DC3000,Pst DC3000)—烟草互作系统为模型,检测了病原菌侵染对烟草褪黑素相关基因表达的影响,并探讨了褪黑素对植物叶片病原菌生长以及气孔开度和活性氧自由基(reactive oxygen species,ROS)含量的影响以及调控机理。结果表明:(1)Pst DC3000处理提高了烟草褪黑素合成(NtSNAT1)和受体(NtPMTR1)基因表达,且外源褪黑素处理降低了叶片中的病原菌含量。(2)与野生型植物相比,过表达大豆GmSNAT1基因显著提高了转基因烟草中内源褪黑素含量和NtPMTR1的表达,且转基因烟草叶片中的Pst DC3000菌落数显著下降。(3)外源褪黑素和细菌鞭毛蛋白多肽flg22处理诱导了野生型和转基因烟草保卫细胞中ROS产生和气孔关闭,且转基因植物对褪黑素和flg22诱导的气孔关闭和ROS产生比野生型烟草更加敏感。综上所述,研究表明褪黑素可能通过受体NtPMTR1介导的信号途径促进保卫细胞ROS产生,诱导气孔关闭,从而降低病原菌Pst DC3000的入侵。     

N-Heterocyclic Carbene-Palladium Polymers Network: Recyclable Pre-catalyst for Effective Suzuki–Miyaura Coupling Reaction in Water

Rational design of high-efficiency N-heterocyclic carbene (NHC) palladium catalyst is of great importance to modern organic synthesis, especially in chemical and pharmaceutical industries. Herein, we fabricate a polymer network containing N-heterocyclic carbene palladium (PNNHC-Pd) catalytic active sites via an immobilization process. The N-heterocyclic carbene palladium can serve as a promising linkage of polymer network as well as an effective catalytic active site owing to its structural preference and strong σ-donating ability with palladium species. The results display that N-heterocyclic carbene palladium disperses homogeneously in polymer network, thus rendering PNNHC-Pd excellent catalytic activity, high stability and superior reusability in palladium-catalyzed Suzuki–Miyaura coupling reaction in aqueous medium. This work provides a new insight into the development of heterogenization of homogeneous catalysts based on polymer network.