Potential of the melanophore pigment response for detection of bacterial toxicity

Chromatophore cells have been investigated as potential biodetectors for function-based detection of chemically and biologically toxic substances. Oncorhynchus tshawytscha (chinook salmon) melanophores, a chromatophore cell type containing brown pigment, rapidly detect the salmonid pathogens Aeromonas salmonicida, Yersinia ruckeri, and Flavobacterium psychrophilum and the human pathogen Bacillus cereus.     

Proximity labeling reveals non-centrosomal microtubule-organizing center components required for microtubule growth and localization

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Evidence for structural changes in carboxyl-terminal peptides of transducin alpha-subunit upon binding a soluble mimic of light-activated rhodopsin

Although a high-resolution crystal structure for the ground state of rhodopsin is now available, portions of the cytoplasmic surface are not well resolved, and the structural basis for the interaction of the cytoplasmic loops with the retinal G-protein transducin (G(t)) is still unknown. Previous efforts aimed at the design, construction, and functional characterization of soluble mimics for the light-activated state of rhodopsin have shown that grafting defined segments from the cytoplasmic region of bovine opsin onto a surface loop in a mutant form of thioredoxin (HPTRX) is sufficient to confer partial G(t) activating potential [Abdulaev et al. (2000) J. Biol. Chem. 275, 39354-39363]. To assess whether these designed mimics could provide a structural insight into the interaction between light-activated rhodopsin and G(t), the ability of an HPTRX fusion protein comprised of the second (CD) and third (EF) cytoplasmic loops (HPTRX/CDEF) to bind G(t) alpha-subunit (G(t)(alpha)) peptides was examined using nuclear magnetic resonance (NMR) spectroscopy. Transfer NOESY (TrNOESY) experiments show that an 11 amino acid peptide corresponding to the carboxyl terminus of G(t)(alpha) (GtP), as well as a "high-affinity" peptide analogue, HAP1, binds to HPTRX/CDEF in the fast-exchange regime and undergoes similar, subtle structural changes at the extreme carboxyl terminus. Observed TrNOEs suggest that both peptides when bound to HPTRX/CDEF adopt a reverse turn that is consistent with the C-cap structure that has been previously reported for the interaction of GtP with the light-activated signaling state, metarhodopsin II (MII). In contrast, TrNOESY spectra provide no evidence for structuring of the amino terminus of either GtP or HAP1 when bound to HPTRX/CDEF, nor do the spectra show any measurable changes in the CD and EF loop resonances of HPTRX/CDEF, which are conformationally dynamic and significantly exchange broadened. Taken together, the NMR observations indicate that HPTRX/CDEF, previously identified as a functional mimic of MII, is also an approximate structural mimic for this light-activated state of rhodopsin.     

Problem of the structural and functional organization of the immature motor center

A hypothetical structural and functional scheme of organization of the immature spinal motor center is proposed, based on our own studies carried out on rat pups of the first month of postnatal development as well as on the analysis of data from literature. Taking into account peculiarities of functioning of various chains of the segmental reflex apparatus (high excitability of the motor center, heterochrony in the development of interneurons of the spinal cord dorsal horns as well as of excitatory and inhibitory mechanisms, possibility of generation of rhythmic activity by one of the half-centers of the motor generator regardless of the activity of the other one) and the mechanisms of its cholinergic and catecholaminergic regulation, age-related changes are considered, which are connected with the organization of interrelations of elements both within the motor center and from the descending regulatory systems.     

Evidence for a rapid structural change in TMV-A-protein near neutrality

Titration of TMV-A-protein from pH 8 to 7 (20°C) or raising the temperature from 4° to 20°C (pH 7) produces, within a few minutes, a reversible change in the aromatic region of the CD-spectrum, before any extensive aggregation has taken place. This spectral change is solely a matter of the conditions of the solution and not of the history of the protein. There is no further CD-change during the slow aggregation process. Thus there must be some proton-uptake within the A-protein. The results are discussed with regard to the different interpretations of the role of A-protein or double-disc in the elongation-step of TMV-“in vitro” -self-assembly.     

A rapid quantitative bioassay for evaluating the effects of ligands upon receptors that modulate cAMP levels in a melanophore cell line.

A new method for rapidly evaluating the effects of drugs on receptors that regulate intracellular cAMP in a cell line derived from Xenopus laevis melanophores has been developed. Melanophores were plated into sterile 96 well microtiter plates, and 3 days later the cells were treated with melatonin for 30 min to induce melanosome aggregation. Subsequent exposure to MSH or adrenergic agonists caused dose dependent pigment dispersion that peaked within 30 min. The cumulative pigment displacement from cells could be quantitated by using a microplate reader to measure changes in transmittance of light through the wells. The acquired data enabled detailed and reproducible dose response curves and time course analyses to be generated. In addition, the assay followed for the rapid characterization of the effects of antagonists upon the beta adrenergic receptor (beta AR). The assay has the potential to test the effects of ligands upon any receptor capable of mediating pigment translocation in the melanophore cell line.     

Taxol affects both the microtubular arrays of heliozoan axonemes and their microtubule-organizing center

Short and long-term effects of the antitumor drug taxol on the microtubular axonemes and on the microtubule-organizing centroplast of the centrolhelidian Heterophrys marina have been investigated. Short-term treatment reveals that general aspects of cell structure remain substantially unaffected and that additional microtubule (MT) assembly in individual axonemes is accompanied by disassembly in others. However, the interaction between MTs, via cross-bridging associated proteins, is seriously affected as indicated by the numerous softly-bent axopods with disturbed arrays of the normally hexagonal pattern. Stabilization of MTs becomes evident by the reexpansion of axopods during low temperature incubation and also by the rapid inhibition of the saltatory movement of the extrusive organelles. Rapidly reexpanding axonemes of cells incubated at higher temperatures and in high taxol concentrations arise asymmetrically from the microtubule-organizing centrosomal structure (centroplast) and form a single thick and thorn-like axopodium, indicating a certain disarrangement of the centrally located microtubule-organizing center (MTOC), which obviously is severely damaged after long-term treatment. With increasing disorganization of the centroplast's structure, these cells reveal themselves unable to sustain their regular microtubular axonemal cytoskeleton. Paradoxically, polymerization of free microtubules from the tubulin pool does not take place. Instead, paracrystalline arrays of twisted filaments appear within the cytoplasm. It is concluded that heliozoan MTs can only persist if stabilized by additional factors, such as permanent interaction with the intact centroplast, and that even in the presence of taxol, MTs unattached to such an MTOC will be intrinsically unstable.     

Evidence of functional and structural changes in the microbial community beneath a succulent invasive plant in coastal dunes

海岸沙地肉质入侵植物对微生物群落功能与结构变化的影响海岸沙地提供了诸多宝贵的生态系统服务,例如耕地保护、水分供应和生物多样性保护,因此海岸沙地代表了重要的栖息地保护区。因为海岸沙地环境恶劣、水资源匮乏、养分贫瘠,所以土壤微生物群落对维持植物多样性至关重要。就海岸沙地的生态系统保护而言,外来入侵植物是主要威胁。本文探讨了沿海地区入侵植物食用日中花(Carpobrotus edulis)对细菌群落功能及结构的各种影响。沙地中土壤有机质含量低,微生物活性有限,所以沙地是一种颇具挑战性的底物。因此,在评估食用日中花对生态系统的影响之前需要对细菌提取进行优化,并开展功能评估。本文首先从群落水平生理特征(CLPP)的角度,比较了采用不同土壤储量、样品量、提取液等12种提取方法对沙地群落功能活性的影响,然后又探讨了食用日中花入侵的沙地菌落功能(利用Biolog Ecoplates)与结构变化(利用PCR-DGGE)。血细胞计数发现盐溶液持续增殖了细菌细胞(P ≤ 0.001)。主成分分析(PCA)结果显示,在有限时间框架(0–24 h)内,碳底物消耗没有显著变化。通过对比天然区与非天然区(种间)及天然区(种内)之间食用日中花入侵后的变化,发现了一种与众不同的碳底物利用模式,食用日中花入侵对群落功能的影响因地而异,而与之相辅相成的是,PCR-DGGE结果表明原沙地的菌落结构不同于食用日中花入侵后沙地的菌落结构。     

Age-related structural and functional changes of brain mitochondria

Normal ageing is associated with a gradual decline in the capacity of various cell types, including neurones, to respond to metabolic stress and return to the resting state. An important factor in the decrease of this 'homeostatic reserve' is the gradual, age-dependent impairment of mitochondrial function. In this article we review some of the major structural and functional changes in mitochondria associated with ageing. Apart from the increased mutations in mitochondrial DNA and the evidence for increased oxidative stress with ageing, we also discuss, in some detail, the importance of the mitochondrial membrane structure and composition (in particular lipid composition) for mitochondrial function in general and during ageing. Although some of the neurodegenerative diseases are also associated with some degree of mitochondrial dysfunction, it is not yet clear if these changes are due to the underlining process of normal, physiological ageing or due to the specific pathophysiologic agents responsible for the neurodegenerative processes. Furthermore, we are proposing that there are important differences between normal ageing and neurodegeneration.     

Evidence for structural symmetry and functional asymmetry in the lactose permease of Escherichia coli

Previous work on the lactose permease of Escherichia coli has shown that mutations along a face of predicted transmembrane segment 8 (TMS-8) play a critical role in conformational changes associated with lactose transport (Green, A. L., and Brooker, R. J. [2001] Biochemistry 40, 12220-12229). Substitutions at positions 261, 265, 268, 272, and 276, which form a continuous stripe along TMS-8, were markedly defective for lactose transport velocity. In the current study, three single mutants (F261D, N272Y, N272L) and a double mutant (T265Y/M276Y) were chosen as parental strains for the isolation of mutants that restored transport function. A total of 68 independent mutants were isolated and sequenced. Forty-four were first-site revertants in which the original mutation was changed back to the wild-type residue or to a residue with a similar side-chain volume. The other 24 mutations were second-site suppressors in TMS-2 (Q60L, Q60P), loop 2/3 (L70H), TMS-7 (V229G/A), TMS-8 (F261L), and TMS-11 (F354V, C355G). On the basis of their locations, the majority of the second-site suppressors can be interpreted as improving the putative TMS-2/TMS-7/TMS-11 interface to compensate for conformational defects imposed by mutations in TMS-8 that disrupt the putative TMS-1/TMS-5/TMS-8 interface. Overall, this paper suggests that the TMS-2/TMS-7/TMS-11 interface is more important from a functional point of view, even though there is compelling evidence for structural symmetry between the two halves of the permease.     

Reactivated melanophore motility: differential regulation and nucleotide requirements of bidirectional pigment granule transport

To study the molecular basis for organized pigment granule transport, procedures were developed to lyse melanophores of Tilapia mossambica under conditions in which pigment granule movements could be reactivated. Gentle lysis of the melanophores resulted in a permeabilized cell model, which, in the absence of exogenous ATP, could undergo multiple rounds of pigment granule aggregation and dispersion when sequentially challenged with epinephrine and cAMP. Both directions of transport required ATP, since aggregation or dispersion in melanophores depleted of nucleotides could be reactivated only upon addition of MgATP or MgATP plus cAMP, respectively. Differences between the nucleotide sensitivities for aggregation and dispersion were demonstrated by observations that aggregation had a lower apparent Km for ATP than did dispersion and could be initiated at a lower ATP concentration. Moreover, aggregation could be initiated by ADP, but only dispersion could be reactivated by the thiophosphate ATP analog, ATP gamma S. The direction of pigment transport was determined solely by cAMP, since pigment granules undergoing dispersion reaggregated when cAMP was removed, and those undergoing aggregation dispersed when cAMP was added. These results provide evidence that pigment granule motility may be based on two distinct mechanisms that are differentially activated and regulated to produce bidirectional movements.     

Evidence for rapid faunal changes on islands in a man-made lake

Summary Few studies of island biogeography have been made on islands in which the time of insularization is precisely known. We tested the effects of island formation on ant species diversity in a man-made lake in South Africa, to determine whether island effects are detectable after only 16 years of insularization. The number of ant species observed at trap-line censuses on islands was significantly correlated with island size (r=0.608; P<0.05) and ant species diversity was generally low compared with similar mainland habitats. Mean species number for all islands, including landbridge islands, was 5.5±3.3 species, and on mainland sites was 7.9±2.85 species. Island effects were more marked on islands <20 ha, which had a mean of 3.3±2.5 species per island. Species number on islands was inversely related to densities of the aggressive Anoplolepis custodiens and A. steingroeveri. These two species were only patchily distributed on mainlands, but these ants were nearly ubiquitous on small islands. Several lines of evidence suggest that this single species domination may be responsible for island effects. Island sites also differed in the number of ant species in different trophic groupings, tending to have fewer granivorous species than the mainland sites, but species in other diet groups were similar in both island and mainland habitats. We conclude that there have been marked changes in the ant faunas on islands smaller than 20 ha apparently due to changes in abundance of the dominant ant species. However, the causes of these changes are unknown.     

Analysis of Tub4p, a yeast gamma-tubulin-like protein: implications for microtubule-organizing center function

gamma-Tubulin is a conserved component of microtubule-organizing centers and is thought to be involved in microtubule nucleation. A recently discovered Saccharomyces cerevisiae gene (TUB4) encodes a tubulin that is related to, but divergent from, gamma-tubulins. TUB4 is essential for cell viability, and epitope-tagged Tub4 protein (Tub4p) is localized to the spindle pole body (Sobel, S.G., and M. Snyder. 1995.J. Cell Biol. 131:1775-1788). We have characterized the expression of TUB4, the association of Tub4p with the spindle pole body, and its role in microtubule organization. Tub4p is a minor protein in the cell, and expression of TUB4 is regulated in a cell cycle-dependent manner. Wild-type Tub4p is localized to the spindle pole body, and a Tub4p- green fluorescent protein fusion is able to associate with a preexisting spindle pole body, suggesting that there is dynamic exchange between cytoplasmic and spindle pole body forms of Tub4p. Perturbation of Tub4p function, either by conditional mutation or by depletion of the protein, results in spindle as well as spindle pole body defects, but does not eliminate the ability of microtubules to regrow from, or remain attached to, the spindle pole body. The spindle pole bodies in tub4 mutant cells duplicate but do not separate, resulting in a monopolar spindle. EM revealed that one spindle pole body of the duplicated pair appears to be defective for the nucleation of microtubules. These results offer insight into the role of gamma- tubulin in microtubule-organizing center function.     

Evidence for structural changes in dermatan sulfate and hyaluronic acid with aging

Three dermatan sulfates (DS18, DS28, and DS35) were isolated from women's skin of ages 19 +/- 2.5, 35 +/- 3.5, 47 +/- 1.7, 60 +/- 0.8, and 75 +/- 5 years. They sequentially precipitated with 18, 28, and 35% ethanol. Their sulfate content was: 23.5, 25.3, and 29.0% (w/w) for DS18 at ages 19-35, 47, and 60 years, respectively; 29.0, 24.0, and 18.8% for DS28; and 18.0, 20.0, and 20.6% for DS35 at ages 19-47, 60, and 75 years, respectively. Both DS18 and DS28 decreased, respectively, from 0.030% (of wet-skin weight) to traces at age 75, and from 0.020 to 0.010% at 60 years. At age 75, DS28 apparently increased by 30%. The DS35 values (traces-0.006%) had no age-related trend. Hyaluronic acid (HA) precipitated with 45% ethanol, was 0.030% of skin-weight at ages 19-47, and decreased to 0.015 and 0.007% at 60 and 75 years, respectively. Its electrophoretic mobility was slower at age 47. In the oldest group, i.r. spectra of HA and DS35 displayed no bands at 1650-1600, 1380, and 1320 cm-1, and a new band at 1560 cm-1. Moreover, ninhydrin-positive material of HA and DS35 increased by 75 and 95%, respectively, and the reducing GlcNAc content of HA decreased. These data showed three chemically different dermatan sulfates (two of which were preponderant) and N-deacetylation of HA and DS35 of the oldest group. After age 47, total DS and HA considerably decreased, DS18 and DS35 were oversulfated, and DS28 became undersulfated with aging.     

Correlation of axenic linkage groups with the position of the microtubule-organizing center in aggregating Dictyostelium.

Positioning of the microtubule-organizing center (MTOC) in Dictyostelium discoideum was found to be genetically regulated. We examined the wild-type strain NC-4 cells independently maintained in different laboratories, freshly recovered cells from spores stocked for over 20 years, the temperature-sensitive growth mutant HU49 isolated from NC-4, as well as strain V-12 which is the opposite mating-type to NC-4. During aggregation on nonnutrient agar plates, all these strains showed similar cell polarity, as defined by the alignment of the nucleus ahead of the MTOC. By contrast, in Ax2 and Ax3, axenic strains carrying axenic mutations on linkage groups II and III, the MTOC was usually positioned ahead of the nucleus. Cells containing axenic linkage group II but not III positioned the MTOC ahead of the nucleus. Conversely cell polarity of strains including axenic linkage group III but not II was similar to that of wild-type cells. Thus axenic linkage group II, probably axeC or other linked gene(s) not yet identified, is responsible for the location of the MTOC anterior to the nucleus during aggregation. The anterior positioning of the MTOCs was prevented by growth on bacteria in cells carrying both axenic linkage groups, but not in those carrying only axenic linkage group II.     

pH-dependent structural changes at the Heme-Copper binuclear center of cytochrome c oxidase

The resonance Raman spectra of the aa3 cytochrome c oxidase from Rhodobacter sphaeroides reveal pH-dependent structural changes in the binuclear site at room temperature. The binuclear site, which is the catalytic center of the enzyme, possesses two conformations at neutral pH, assessed from their distinctly different Fe-CO stretching modes in the resonance Raman spectra of the CO complex of the fully reduced enzyme. The two conformations (alpha and beta) interconvert reversibly in the pH 6-9 range with a pKa of 7.4, consistent with Fourier transform infrared spectroscopy measurements done at cryogenic temperatures (D.M. Mitchell, J.P. Sapleigh, A.M.Archer, J.O. Alben, and R.B.Gennis, 1996, Biochemistry 35:9446-9450). It is postulated that the different structures result from a change in the position of the Cu(B) atom with respect to the CO due to the presence of one or more ionizable groups in the vicinity of the binuclear center. The conserved tyrosine residue (Tyr-288 in R. sphaeroides, Tyr-244 in the bovine enzyme) that is adjacent to the oxygen-binding pocket or one of the histidines that coordinate Cu(B) are possible candidates. The existence of an equilibrium between the two conformers at physiological pH and room temperature suggests that the conformers may be functionally involved in enzymatic activity.