Visual accommodation in vertebrates: mechanisms, physiological response and stimuli

The mechanism and stimulation of the accommodative reflex in vertebrate eyes are reviewed. Except for lampreys, accommodation is brought about by intraocular muscles that mediate either a displacement or deformation of the lens, a change of the corneal radius of curvature or a combination of these mechanisms. Elasmobranchs have little accommodation and are emmetropic in water rather than hyperopic as commonly stated. Accommodation in teleosts and amphibians is well understood and achieved by lens displacement. The accommodative mechanism of amniotes is of considerable diversity and reflects different lifestyles rather than phylogenetical relationships. In all amniotes, the ciliary muscle never has a direct impact on the lens. It relaxes the tension applied to the lens by zonular fibers and/or ligaments. In birds and reptiles the ciliary muscle is usually split into two parts, of which the anterior portion changes the corneal radius of curvature. The deformation of the lens is generally achieved either by its own elasticity (humans, probably other mammals and sauropsids) or by the force of circular muscle fibers in the iris (reptiles, birds, aquatic mammals). In the second part of the paper, some of the current hypotheses about the accommodative stimulus are reviewed together with physiological response characteristics.     

Cellular and biochemical mechanisms underlying circadian rhythms in vertebrates

Circadian clocks organize neural processes, such as motor activities, into near 24-hour oscillations and adaptively synchronize these rhythms to the solar cycle. Recently, the first mammalian clock genes have been found. Unpredicted diversity in signaling pathways and clock-controlled gating of signals that modulate timekeeping has been discovered. A diffusible clock output has been found to control some behavioral rhythms. Consensus is emerging that circadian mechanisms are conserved across phylogeny, but that mammals have developed a great complexity of controls.     

Small heat-shock proteins function in the insoluble protein complex

Small heat-shock proteins (sHSPs) represent an abundant and ubiquitous family of molecular chaperones. The current model proposes that sHSPs function to prevent irreversible aggregation of non-native proteins by forming soluble complex. The chaperone activity of sHSPs is usually determined by the capacity to suppress thermally or chemically induced protein aggregation. However, sHSPs were frequently found in the insoluble complex particularly in vivo. In this report, it is clearly revealed that the insoluble sHSP/substrate complex is formed when sHSP is overloaded with non-native substrates, which is the very case under in vivo conditions. The proposal that sHSPs function to prevent the protein aggregation seems misleading. sHSPs appear to promote the elimination of protein aggregates by incorporating into the insoluble protein complex.     

Lens protein expression in mammals: Taxon-specificity and the recruitment of crystallins

Summary Vertebrate lenses show remarkably taxon-specific patterns of protein composition, most obviously in the recruitment of enzymes as major crystallins. Phylogenetic relationships are particularly apparent in mammals. Here we describe ν-crystallin, which is probably identical to cytosolic aldehyde dehydrogenase, lens-specifically expressed at high abundance in the elephant shrews, primitive eutherians of the family Macroscelidae, and μ-crystallin, a novel lens protein expressed in some marsupials. We have also observed that enzymes that have been recruited as crystallins in some species are also moderately abundant in the lenses of other species. This hints that the origins of enzyme-crystallins may lie in a pool of enzymes widely expressed in lenses at fairly high levels, perhaps because they have important developmental or functional roles in the tissue.     

包涵体蛋白的层析复性技术研究进展

包涵体蛋白的复性是生物工程下游技术中的一个重要难题。层析法用于蛋白质复性是一种较新的、适用于大多数蛋白的方法。其原理是将层析技术应用于蛋白质复性和纯化,使变性蛋白质在层析柱上重折叠为正确的空间构象,在洗脱的同时实现部分纯化。本文详细介绍了蛋白质在5种层析柱上的复性方法、原理、应用及研究的新进展,为层析法对蛋白质复性的进一步应用提供依据。     

Tempo and mode of mitochondrial DNA evolution in vertebrates at the amino acid sequence level: Rapid evolution in warm-blooded vertebrates

Summary By using complete sequence data of mitochondrial DNAs, three Markov models (Day-hoff, Proportional, and Poisson models) for amino acid substitutions during evolution were applied in maximum likelihood analyses of mitochondrially encoded proteins to estimate a phylogenetic tree depicting human, cow, whale, and murids (mouse and rat), with chicken, frog, and carp as outgroups. A cow/whale clade was confirmed with a more than 99.8% confidence level by any of the three models, but the branching order among human, murids, and the cow/whale clade remained uncertain. It turned out that the Dayhoff model is by far the most appropriate model among the alternatives in approximating the amino acid substitutions of mitochondrially encoded proteins, which is consistent with a previous analysis of a more limited data set. It was shown that the substitution rate of mitochondrially encoded proteins has increased in the order of fishes, amphibians, birds, and mammals and that the rate in mammals is at least six times, probably an order of magnitude, higher than that in fishes. The higher evolutionary rate in birds and mammals than in amphibians and fishes was attributed to relaxation of selective constraints operating on proteins in warm-blooded vertebrates and to high mutation rate of bird and mammalian mitochondrial DNAs.Offprint requests to: M. Hasegawa     

Procedures for the biochemical enrichment and proteomic analysis of the cytoskeletome

The cell cytoskeleton is composed of microtubules, intermediate filaments, and actin that provide a rigid support structure important for cell shape. However, it is also a dynamic signaling scaffold that receives and transmits complex mechanosensing stimuli that regulate normal physiological and aberrant pathophysiological processes. Studying cytoskeletal functions in the cytoskeleton’s native state is inherently difficult due to its rigid and insoluble nature. This has severely limited detailed proteomic analyses of the complex protein networks that regulate the cytoskeleton. Here, we describe a purification method that enriches for the cytoskeleton and its associated proteins in their native state that is also compatible with current mass spectrometry-based protein detection methods. This method can be used for biochemical, fluorescence, and large-scale proteomic analyses of numerous cell types. Using this approach, 2346 proteins were identified in the cytoskeletal fraction of purified mouse embryonic fibroblasts, of which 635 proteins were either known cytoskeleton proteins or cytoskeleton-interacting proteins. Functional annotation and network analyses using the Ingenuity Knowledge Database of the cytoskeletome revealed important nodes of interconnectivity surrounding well-established regulators of the actin cytoskeleton and focal adhesion complexes. This improved cytoskeleton purification method will aid our understanding of how the cytoskeleton controls normal and diseased cell functions.     

Bone Morphogenetic Protein functions as a context-dependent angiogenic cue in vertebrates

Bone Morphogenetic Protein (BMP) signaling has been implicated in diverse biological processes. Although how BMP signaling regulates behaviors of endothelial cells during angiogenesis are not fully understood, increasing evidence indicate functions of BMP signaling components are essential in developmental and pathological angiogenesis. Here we review recent advances in delineating the functions of BMP signaling during angiogenesis. In addition, we discuss downstream pathways that transduce BMP signaling in endothelial cells, and factors that modulate BMP signaling response in endothelial cells. Finally, we provide recent insight on how BMP signaling functions as a context dependent angiogenic cue.     

Interactions of lens proteins. Ultrafiltration is unsuitable to detect self- or mixed-association

Crystallins from calf lens were subjected to ultrafiltration through an Amicon XM-300 membrane to determine whether specific interactions between identical proteins (self-association) or different proteins (mixed-association) could be detected and quantified. Single crystallins at different concentrations, simple mixtures and total lens extracts were studied separately. alpha-Crystallin (Mr 800 000) is nearly fully retained (greater than 95%) by XM-300. Retention of beta-crystallins (Mr 50 000-200 000) is found to be much higher than expected from their molecular weights. Ultrafiltration of gamma-crystallin (Mr 20 000) solutions of 1.0-22.6 g/l shows that retention increases as a function of protein concentration. In solutions of single crystallins, self-association effects could not be separated from concentration polarization effects at the membrane surface. In mixtures of crystallins, mixed-association could not be separated from self-association, concentration polarization and excluded volume effects on self-association.     

Changes in protein synthesis during the initial stage of life of tobacco protoplasts

The biosynthesis of Fraction I protein in isolated protoplasts is compared with that in the plant. Radioactive precursors were incorporated into isolated protoplasts (in vitro labeling) and into leaves, from which the protoplasts were isolated later (in situ labeling). The biosynthesis of Fraction I protein stopped almost completely as soon as the protoplasts were incubated in the culture medium.     

The common modification in alphaA-crystallin in the lens, N101D, is associated with increased opacity in a mouse model

To elucidate the morphological and cellular changes due to introduction of a charge during development and the possible mechanism that underlies cataract development in humans as a consequence of an additional charge, we generated a transgenic mouse model mimicking deamidation of Asn at position 101. The mouse model expresses a human αA-crystallin gene in which Asn-101 was replaced with Asp, which is referred to as αAN101D-transgene and is considered to be "deamidated" in this study. Mice expressing αAN101D-transgene are referred to here CRYAA(N101D) mice. All of the lines showed the expression of αAN101D-transgene. Compared with the lenses of mice expressing wild-type (WT) αA-transgene (referred to as CRYAA(WT) mice), the lenses of CRYAA(N101D) mice showed (a) altered αA-crystallin membrane protein (aquaporin-0 (AQP0), a specific lens membrane protein) interaction, (b) extracellular spaces between outer cortical fiber cells, (c) attenuated denucleation during confocal microscopic examination, (d) disrupted normal fiber cell organization and structure during scanning electron microscopic examination, (e) distorted posterior suture lines by bright field microscopy, and (f) development of a mild anterior lens opacity in the superior cortical region during the optical coherence tomography scan analysis. Relative to lenses with WT αA-crystallin, the lenses containing the deamidated αA-crystallin also showed an aggregation of αA-crystallin and a higher level of water-insoluble proteins, suggesting that the morphological and cellular changes in these lenses are due to the N101D mutation. This study provides evidence for the first time that expression of deamidated αA-crystallin caused disruption of fiber cell structural integrity, protein aggregation, insolubilization, and mild cortical lens opacity.     

The golden age of biochemical research in photosynthesis

The perspectives and enthusiasms recorded in this review describe the events I witnessed and, in small ways, contributed to. Two great rewards emerged from my experiences – the pleasure of doing experiments and the great wealth of friendships with students and colleagues. As a graduate student, phenomena appeared at the bench before me which clarified the coupling of electron transport to ATP synthesis. My first PhD graduate student measured concentrations of pyridine nucleotides in chloroplasts and his results have been often confirmed and well used. All of the many graduate students who followed contributed to our understanding of photosynthesis. I have taken much pleasure from documenting the details of photosynthetic phosphorylation and electron transport in cyanobacteria. Studies of the `c' type cytochromes in these organisms continue to fascinate me. My experiences in government in its efforts to promote research are unusual, perhaps unique. A rare event outside the laboratory – a natural bloom of cyanobacteria – stimulated new thoughts and special opportunities for laboratory science. Photosynthesis seems magisterial in its shaping of our planet and its biology and in the details of its cleverness that were revealed in the time of my witness. This revised version was published online in June 2006 with corrections to the Cover Date.     

A biochemical characterization of the photophore lenses of the midshipman fish,Porichthys notatus Girard

The present study is a biochemical characterization of the photophore lenses of the midshipman fish, Porichthys notatus, a species that bears 800 photophores distributed over the body surface. The biochemical properties of the photophore lenses were compared with those of the eye lens with which they share a similar developmental origin and analogous function. To achieve a high refractive index, the vertebrate eye lens has a relatively high concentration of structural proteins (20–50%, depending on species) and a simple protein composition, that is, relatively few proteins are synthesized in comparison to other tissues. Similarly, the photophore lenses of P. notatus had a relatively high protein concentration (average = 29%, n = 5) and approximately 60% of the total soluble protein was represented by two subunit species of 33 kD and 35 kD on denaturing polyacrylamide gels. The structural proteins of the eye lens are of two principle types: 1) and polypeptides which belong to vertebrate lens-specific crystallin families, and, 2) enzymes recruited into the lens which take on the function of structural proteins. Here, we report that the two major photophore lens subunits of 33 kD and 35 kD are biochemically similar to each other, but are clearly distinct from any of the previously characterized crystallins. Therefore, we propose that photophore lenses appear to recruit a novel protein.     

Structural and biochemical characterization of the salicylyl-acyltranferase SsfX3 from a tetracycline biosynthetic pathway

SsfX3 is a GDSL family acyltransferase that transfers salicylate to the C-4 hydroxyl of a tetracycline intermediate in the penultimate step during biosynthesis of the anticancer natural product SF2575. The C-4 salicylate takes the place of the more common C-4 dimethylamine functionality, making SsfX3 the first acyltransferase identified to act on a tetracycline substrate. The crystal structure of SsfX3 was determined at 2.5 Å, revealing two distinct domains as follows: an N-terminal β-sandwich domain that resembles a carbohydrate-binding module, and a C-terminal catalytic domain that contains the atypical α/β-hydrolase fold found in the GDSL hydrolase family of enzymes. The active site lies at one end of a large open binding pocket, which is spatially defined by structural elements from both the N- and C-terminal domains. Mutational analysis in the putative substrate binding pocket identified residues from both domains that are important for binding the acyl donor and acceptor. Furthermore, removal of the N-terminal carbohydrate-binding module-like domain rendered the stand-alone α/β-hydrolase domain inactive. The additional noncatalytic module is therefore proposed to be required to define the binding pocket and provide sufficient interactions with the spatially extended tetracyclic substrate. SsfX3 was also demonstrated to accept a variety of non-native acyl groups. This relaxed substrate specificity toward the acyl donor allowed the chemoenzymatic biosynthesis of C-4-modified analogs of the immediate precursor to the bioactive SF2575; these were used to assay the structure activity relationships at the C-4 position.     

A test of the lipofuscin technique of age estimation in fish

A stable extraction technique was developed to quantify the lipofuscin content of fish brains. Samples of brains of the same age but different size rass carp showed that a large part of the variation in lipofuscin content was related to the weight of the fish.     

Changing protein patterns during lens cell aging in vitro

Changes in biosynthesis of lens proteins upon culturing have been studied by one- and two-dimensional gel electrophoretic techniques. In primary cells still growing on the capsule, αB₂-crystallin is synthesized in a relatively high amount next to the main cytoskeletal constituents actin, tubulin and vimentin. In addition, a minor amount of βB_p seems to be synthesized too. When the cells grow off the capsule, α-crystallin synthesis diminishes. β-Crystallin synthesis continues at a low rate in cells growing on plastic or in cells forming ‘lentoid bodies’. When the cells are subcultured, the synthesis of actin and vimentin becomes more pronounced, while tubulin synthesis is no longer detectable after three transfes The relative amount of vimentin decreases, as compared to actin, during aging and elongation of the cells. When the cells have been transferred ten times and have started to elongate, a 55 kDa protein doublet differing from tubulin is observed in the two-dimensional gel patterns. We observed that elongation of lens cells in culture is accompanied by an increase in the synthesis of a polypeptide of the 26 kDa region. Furthermore, a major glycoprotein is found in the 130 kDa region, but overall glycosylation of proteins seems to decrease during lens cell elongation in vitro.     

The European Proteomics Association (EuPA) is in the field. Report of the formal inauguration of the European Proteome Association (Munich, Germany, August 29, 2005)

After one year of preparation the European Proteomics Association (EuPA) was formally inaugurated on August 29, 2005, on the occasion of the 4(th) HUPO World Congress in Munich, Germany. Delegates from 16 European countries elected Dr. Friedrich Lottspeich, President of the German Proteome Society, as the first EuPA President. The EuPA Board also comprises Professor Mathias Uhlen as Vice President, along with Professor Michael Dunn (Ireland), Professor Concha Gil (Spain), Dr. Jean Charles Sanchez (Switzerland) and Professor Pier Giorgio Righetti (Italy) as Coordinators for the presently defined focus activities of the EuPA. The general objectives of the EuPA are to promote proteomic activities throughout Europe, emphasising the benefits and contribution of proteomics to biological researchers, industry, the general public and politicians.     

Incremental lines of dental cementum in biological age estimation

The aim of this study is to evaluate the use of counting incremental lines of dental root cementum for biological age determination, and to compare it with alternative methods. Two samples were taken: 51 teeth from 49 individuals of known age obtained at the Stomatological Clinic, Vilnius University, as well as the canine teeth from the remains of 48 individuals from the mass graves of Tuskulenai in Vilnius (inhumed 1944-47). In the latter sample, the chronological age of 43 individuals was known through personal identification. Undecalcified teeth were sectioned with the Leica SP 1600 microtome diamond saw, and incremental line count as a blind test was made on sections of 35 to 100 microns thickness. Incremental line count was possible in 82-86 percent of cases. The results of three independent counts showed that intra-observer bias has no significant impact. Biological age was estimated by adding incremental line number to the average age of tooth eruption. It was found that mean absolute error was 6.46 years for the 1st sample, 6.27 years for the 2nd sample, and in some cases exceeded 10 years. For the 2nd sample, the results were compared to those of other methods such as endocranial suture ossification, pubic symphysis morphology and the "combined" method of Nemeskéri. All four methods yield a similar correlation in regard to an individual's chronological age. The highest correlation was found for the combined method, and the lowest one for public symphysis morphology. All correlations had a similar standard error. Thus our assessment is less enthusiastic than in some past studies; it is suggested that the incremental lines rather have a similar use as other methods.