Bend propagation drives central pair rotation in Chlamydomonas reinhardtii flagella

Regulation of motile 9+2 cilia and flagella depends on interactions between radial spokes and a central pair apparatus. Although the central pair rotates during bend propagation in flagella of many organisms and rotation correlates with a twisted central pair structure, propulsive forces for central pair rotation and twist are unknown. Here we compared central pair conformation in straight, quiescent flagella to that in actively beating flagella using wild-type Chlamydomonas reinhardtii and mutants that lack radial spoke heads. Twists occur in quiescent flagella in both the presence and absence of spoke heads, indicating that spoke--central pair interactions are not needed to generate torque for twisting. Central pair orientation in propagating bends was also similar in wild type and spoke head mutant strains, thus orientation is a passive response to bend formation. These results indicate that bend propagation drives central pair rotation and suggest that dynein regulation by central pair--radial spoke interactions involves passive central pair reorientation to changes in bend plane.     

昆虫嗅觉相关蛋白的研究进展

嗅觉是昆虫产生行为的重要物质基础,阐明昆虫嗅觉机理有助于调控昆虫行为和进行害虫治理。近年来,许多与嗅觉相关的生物活性分子和相关基因的发现和克隆,对揭示嗅觉机理具有重要作用。作者针对近年来研究较多的气味结合蛋白、化学感受蛋白、气味受体、气味降解酶以及感觉神经元膜蛋白等,就其生化特性、表达部位、分子结构、生理功能等进行了综述。     

Mitosis and microtubule organizational changes in rice root-tip cells

The pattern of change of the microtubule cytoskeleton of the root-tip cells of rice during mitosis was studied using immunofluorescence technic and confocal laser scanning microscopy. All the major stages of ceil division including preprophase, prophase, metaphase, anaphase and telophase were observed. The most significant finding was that in the preprophase cells microtubules radiating from the nuclear surface to the cortex were frequently seen. During development these microtubules became closely associated with the preprophase band and prophase spindie indicating that the microtubules radiating from the nuclear surface, the preprophase band and the prophazc spindle were structurally and functionally closely related to each other. Granule-like anchorage sites for the radiating microtubules at the muclear surface were often seen and the possibility that these gramle-like anchorage sites might represent the microtubule organizing centres was discussed.     

S-Carboxymethylcysteine Synthase from Escherichia coli

An enzyme that catalyzes the synthesis of S-carboxymethyl- l-cysteine from 3-chloro- l-alanine (3-Cl-Ala) and thioglycolic acid was found in Escherichia coli W3110 and was designated as S- carboxymethyl-l-cysteine synthase. It was purified from the cell-free extract to electrophoretic homogeneity and was crystallized. The enzyme has a molecular weight of 84,000 and gave one band corresponding to a molecular weight of 37,000 on SDS-polyacrylamide gel electrophoresis. The purified enzyme catalyzed the β-replacement reactions between 3-CI-AIa and various thiol compounds. The apparent Km values for 3-Cl-Ala and thioglycolic acid were 40 mM and 15.4 mM. The enzyme showed very low activity as to the α,β-elimination reaction with 3-Cl-Ala and l-serine. It was not inactivated on the incubation with 3-Cl-Ala. The absorption spectrum of the enzyme shows a maximum at 412 nm, indicating that it contains pyridoxal phosphate as a cofactor. The N-terminal amino acid sequence was determined and the corresponding sequence was detected in the protein sequence data bank, but no homogeneous sequence was found.     

Electrogenic Properties of the Sodium Pump in a Dynamic Model of Membrane Transport

The general purpose of this theoretical work is to contribute to understand the physiological role of the electrogenic properties of the sodium pump, by studying a dynamic model that integrates diverse processes of ionic and water transport across the plasma membrane. For this purpose, we employ a mathematical model that describes the rate of change of the intracellular concentrations of Na⁺, K⁺ and Cl⁻, of the cell volume, and of the plasma membrane potential (V _m ). We consider the case of a nonexcitable, nonpolarized cell expressing the sodium pump; Na⁺, K⁺, Cl⁻ and water channels, and cotransporters of KCl and NaCl in its plasma membrane. We particularly analyze here the conditions under which the physiological V _m can be generated in a predominantly electrogenic fashion, as a result of the activity of the sodium pump. A major conclusion of this study is that, for the cell model considered, a low potassium permeability is not a sufficient condition for a predominantly electrogenic generation of the V _m by the sodium pump. The presence of an electroneutral exchange of Na⁺ and K⁺ represents a necessary additional requirement. Received: 8 September 1999/Revised: 21 March 2000     

Differences in the chitinolytic activity of mammalian chitinases on soluble and insoluble substrates

Chitin is an abundant polysaccharide used by many organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Acidic mammalian chitinase, a processive glycosyl hydrolase, is the primary enzyme involved in the degradation of environmental chitin in mammalian lungs. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion in mice increases morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a screening approach with commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with insoluble chitin, and identified a one‐pot fluorogenic assay that is sufficiently sensitive to quantify changes to activity due to the addition or removal of a carbohydrate‐binding domain. We show that the activity increases from our directed evolution screen were lost when insoluble substrates were used. In contrast, naturally occurring gain‐of‐function mutations gave similar results with oligomeric and insoluble substrates. We also show that activity differences between acidic mammalian chitinase and chitotriosidase are reduced with insoluble substrate, suggesting that previously reported activity differences with oligomeric substrates may have been driven by differential substrate specificity. These results highlight the need for assays against physiological substrates when engineering metabolic enzymes, and provide a new one‐pot assay that may prove to be broadly applicable to engineering glycosyl hydrolases.     

Metal accumulation and sublethal effects in the sea anemone, Aiptasia pallida, after waterborne exposure to metal mixtures

The marine environment is subjected to contamination by a complex mixture of metals from various anthropogenic sources. Measuring the biological responses of organisms to a complex mixture of metals allows for examination of metal-specific responses in an environmentally realistic exposure scenario. To address this issue, the sea anemone, Aiptasia pallida was exposed to a control and a metal mixture (copper, zinc, nickel, and cadmium) at three exposure levels (10, 50, and 100 μg/L) for 7 days. Anemones were then transferred to metal-free seawater for an additional 7 days after the metal exposure to assess metal depuration and recovery. Metal accumulation, activity of the enzymes catalase, glutathione reductase, and carbonic anhydrase, as well as, cell density of the symbiotic zooxanthellae were measured over 14 days. Metal accumulation in A. pallida occurred in a concentration dependent manner over the 7-day exposure period. Altered enzyme activity and tentacle retraction of the host, as well as decreased zooxanthellae cell density were observed responses over the 7 days, after exposure to a metal concentration as low as 10 μg/L. Metal depuration and physiological recovery were dependent on both the metal and the exposure concentration. Understanding how A. pallida and their symbionts are affected by metal exposures in the laboratory may allow better understanding about the responses of symbiotic cnidarians in metal polluted aquatic environments.     

Extracellular peptidases of insect-associated fungi and their possible use in biological control programs and as pathogenicity markers

This review deals with characteristics of peptidases of fungi whose life cycles are associated with insects to varying degrees. The review examines the characteristic features of the extracellular peptidases of entomopathogenic fungi, the dependence of the specificity of these peptidases on the ecological characteristics of the fungi, and the role of peptidases in the development of the pathogenesis. Data on the properties and physiological role of hydrolytic enzymes of symbiotic fungi in “fungal gardens” are also considered in detail. For the development of representations about mechanisms of control over populations of insect pests, special attention is given to analysis of possibilities of genetic engineering for the creation of entomopathogens with enhanced virulence. Clarification of the role of fungi and their secreted enzymes and careful environmental studies are still required to explain their significance in the composition of the biota and to ensure widespread adoption of these organisms as effective biological control agents. The systematization and comparative analysis of the existing data on extracellular peptidases of insect-associated fungi will help in the planning of further work and the search for markers of pathogenesis and symbiosis.