Structures and functions of the sugar chains of glycoproteins.

Most proteins within living organisms contain sugar chains. Recent advancements in cell biology have revealed that many of these sugar chains play important roles as signals for cell-surface recognition phenomena in multi-cellular organisms. In order to elucidate the biological information included in the sugar chains and link them with biology, a novel scientific field called 'glycobiology' has been established. This review will give an outline of the analytical techniques for the structural study of the sugar chains of glycoproteins, the structural characteristics of the sugar chains and the biosynthetic mechanism to produce such characteristics. Based on this knowledge, functional aspects of the sugar chains of glycohormones and of those in the immune system will be described to help others understand this new scientific field.     

Longevity control in fungi and other organisms. The conception of scales

The review deals mainly with gerontological processes that occur on the cellular-colonial level of organization in fungi and cellular-tissular level in other organisms. Aging and anti-aging mechanisms operating on these levels of organization can be considered as common ones for all living things. Fungi, as an object with tissular-like organization of thallus, afford a broad spectrum of possibilities as to solving the tasks of general gerontological import. Three basic (chronological, replicative, and cell-suicidal) and several auxiliary mechanisms of aging are singled out, the classification is given of stochastic aging factors accumulating in cells. It is shown that in complex multi-cellular organisms, aging and anti-aging mechanisms operate on the level of interactions between tissues, though in the base of their actions lie the aforesaid conservative basic mechanisms. Preliminary generalized conception of aging--the conception of scales--is put forward that is founded on the model of balanced and non-balanced counteractions between stressful impacts and various mechanisms of aging and anti-aging with different extent of genetic preprogramming. The importance is reaffirmed of mycological gerontology contribution to broadening of inferences on aging nature.     

Late-Onset of Spinal Neurodegeneration in Knock-In Mice Expressing a Mutant BiP

Most human neurodegenerative diseases are sporadic, and appear later in life. While the underlying mechanisms of the progression of those diseases are still unclear, investigations into the familial forms of comparable diseases suggest that endoplasmic reticulum (ER) stress is involved in the pathogenesis. Binding immunoglobulin protein (BiP) is an ER chaperone that is central to ER function. We produced knock-in mice expressing a mutant BiP that lacked the retrieval sequence in order to evaluate the effect of a functional defect in an ER chaperone in multi-cellular organisms. Here we report that heterozygous mutant BiP mice revealed motor disabilities in aging. We found a degeneration of some motoneurons in the spinal cord accompanied by accumulations of ubiquitinated proteins. The defect in retrieval of BiP by the KDEL receptor leads to impaired activities in quality control and autophagy, suggesting that functional defects in the ER chaperones may contribute to the late onset of neurodegenerative diseases.     

Programmed longevity,youthspan, and juventology

The identification of conserved genes and pathways that regulate lifespan but also healthspan has resulted in an improved understanding of the link between nutrients, signal transduction proteins, and aging but has also provided evidence for the existence of multiple “longevity programs,” which are selected based on the availability of nutrients. Periodic fasting and other dietary restrictions can promote entry into a long‐lasting longevity program characterized by cellular protection and optimal function but can also activate regenerative processes that lead to rejuvenation, which are independent of the aging rate preceding the restricted period. Thus, a “juventology”‐based strategy can complement the traditional gerontology approach by focusing not on aging but on the longevity program affecting the life history period in which mortality is very low and organisms remain youthful, healthy, and fully functional.     

Comparative study of the asparagine-linked sugar chains of human lipocalin-type prostaglandin D synthase purified from urine and amniotic fluid, and recombinantly expressed in Chinese hamster ovary cells

Lipocalin-type prostaglandin D synthase (L-PGDS) is a highly glycosylated member of the lipocalin gene family and is secreted into various human body fluids. We comparatively analyzed the structures of asparagine-linked sugar chains of human L-PGDS produced by recombinant Chinese hamster ovary cells and naturally occurring human urine and amniotic fluid. After the sugar chains were liberated by hydrazinolysis followed by N-acetylation, they were derivatized with 2-aminobenzamide. All of the sugar chains of three L-PGDSs occur as biantennary complex-type sugar chains. Most of the sugar chains of three samples were fucosylated on the inner most N-acetylglucosamine residue. Although the sugar chains of the recombinant L-PGDS do not contain any bisecting N-acetylglucosamine residues, 58% and 34% of the fucosylated-sugar chains of amniotic fluid and urine L-PGDSs, respectively, contain bisecting N-acetylglucosamine residues. The sialic acid residues occur solely as Siaalpha2-->3Gal groups of the recombinant L-PGDS; the sialic acid residues of other L-PGDS occur as both Siaalpha2-->3Gal and Siaalpha2-->6Gal groups. Variations in L-PGDS glycosylation may prove useful as markers to further elucidate the role of L-PGDS glycoforms in different tissues.     

Comparative study of the asparagine-linked sugar chains of natural human interferon-beta 1 and recombinant human interferon-beta 1 produced by three different mammalian cells

The asparagine-linked sugar chains of natural interferon-beta 1 secreted from human foreskin fibroblasts by poly I:poly C induction and of three recombinant human interferon-beta 1 produced by Chinese hamster ovary cells, mouse epithelial cells (C127), and human lung adenocarcinoma cells (PC8) were released quantitatively as oligosaccharides by hydrazinolysis followed by N-acetylation. After being reduced with either NaB3H4 or NaB2H4, their structures were comparatively analyzed. More than 80% of the sugar chains of natural interferon-beta 1 occur as biantennary complex-type sugar chains, approximately 10% of which contain N-acetyllactosamine repeating structure in their outer chain moieties. The remainders are 2,4- and 2,6-branched triantennary complex-type sugar chains. The sugar chains of the recombinant interferon-beta 1 derived from Chinese hamster ovary cells were very similar to those of its natural counterpart. In contrast, two other recombinant proteins contain quite different sugar chains. The protein derived from C127 cells contains complex-type sugar chains with the Gal alpha 1----3Gal beta 1----4GlcNAc group in their outer chain moieties. Their sialic acid residues occur solely as the Sia alpha 2----6Gal group, where Sia is sialic acid. In contrast, the sialic acid residues of other interferon-beta 1 occur as the Sia alpha 2----3Gal group only. A part of the sugar chains of the protein derived from PC8 cells contains bisecting N-acetylglucosamine residue in addition to the Gal alpha 1----3Gal beta 1----4GlcNAc group.     

昆虫飞行肌细胞凋亡的分子生物学研究进展

细胞凋亡是多细胞生物体内由激素刺激,基因调控,蛋白调节的一个主动的程序化死亡过程,对生物体特定组织功能的发生、发展、衰老及退化等具有重要作用。而昆虫飞行肌细胞凋亡对昆虫迁飞行为尤为重要,它直接决定迁飞性昆虫能否选择更适宜的寄主植物和生活条件,进而影响到对农作物的危害地域和严重程度。本文在近年来昆虫细胞凋亡的研究基础上,从分子生物学的角度,综述了调控昆虫飞行肌细胞凋亡的相关基因和蛋白质的研究进展,探讨了昆虫飞行肌细胞凋亡发生与调控的分子生物学机制。     

丝状真菌糖生物学

蛋白质的糖基化修饰是一种保守的真核生物蛋白质翻译后修饰,存在于从酵母到人的所有真核生物中,赋予了蛋白质功能的多样性。目前对蛋白质糖基化修饰的了解主要来源于对酵母和哺乳动物细胞的研究,但单细胞真核生物或动物细胞水平的研究,很难反映糖基化修饰在多细胞真核生物的发育分化过程中的复杂功能。由于丝状真菌是多细胞真核生物,有相对简单的发育分化过程,因而是研究多细胞真核生物糖基化功能的理想模型之一,在过去10年中,丝状真菌的糖生物学研究开始受到重视,目前的研究结果表明糖基化修饰与丝状真菌的生长、发育密切相关。     

Caenorhabditis elegans DNA-2 helicase/endonuclease plays a vital role in maintaining genome stability, morphogenesis, and life span

In eukaryotes, highly conserved Dna2 helicase/endonuclease proteins are involved in DNA replication, DNA double-strand break repair, telomere regulation, and mitochondrial function. The Dna2 protein assists Fen1 (Flap structure-specific endonuclease 1) protein in the maturation of Okazaki fragments. In yeast, Dna2 is absolutely essential for viability, whereas Fen1 is not. In Caenorhabditis elegans, however, CRN-1 (a Fen1 homolog) is essential, but Dna2 is not. Here we explored the biological function of C. elegans Dna2 (Cedna-2) in multiple developmental processes. We find that Cedna-2 contributes to embryonic viability, the morphogenesis of both late-stage embryos and male sensory rays, and normal life span. Our results support a model whereby CeDNA-2 minimizes genetic defects and maintains genome integrity during cell division and DNA replication. These finding may provide insight into the role of Dna2 in other multi-cellular organisms, including humans, and could have important implications for development and treatment of human conditions linked to the accumulation of genetic defects, such as cancer or aging.     

Genetic and evolutionary aspects of aging.

Four questions of fundamental importance to gerontology are considered. 1) The number of genes involved in aging--in the case of man, an analysis of the phenotypes of relevant spontaneous mutants indicates that aging is highly polygenic. 2) General categories of genes--regulator genes may be more relevant than structural genes: a) three aneuploid disorders, Down's, Turner's and Klinefelter's syndromes, ranked among the top 10 candidates as "segmental progeroid syndromes" when compared with 162 single gene disorders of relevance to the pathobiology of aging; b) the rates at which maximum life spans have been increasing, especially among hominids, have probably been too rapid to be accounted for by changes in the amino acid sequences of proteins; c) a preliminary analysis of the variance of maximum life spans among a few orders of mammals is suggestive of a linear correlation with the indexes of rates of chromosomal evolution, as estimated by Bush et al. (Proc. Natl. Acad. Sci. USA 74: 3942-3946, 1977). 3) Nature of gene action--although there are reasons for invoking genes that modulate the rates of accumulation of somatic mutations, differential regulation of development is likely to be a major setting for gene action. 4) New approaches to formal genetic analysis of aging--advances in experimental embryology and somatic cell genetics offer such opportunities.     

Carbohydrate structures of human interleukin 5 expressed in Chinese hamster ovary cells.

The asparagine-linked sugar chains of recombinant human interleukin 5 produced by Chinese hamster ovary cells were released quantitatively as oligosaccharides by hydrazinolysis. After N-acetylation followed by NaB3H4 reduction, each oligosaccharide was isolated by paper electrophoresis and serial lectin column chromatography. Study of their structures by sequential exoglycosidase digestion in combination with methylation analysis, revealed that they are bi-, tri-, and tetraantennary complex-type with fucosylated and non-fucosylated trimannosyl cores and high mannose type sugar chains. More than 80% of the sugar chains occur as biantennary complex-type sugar chains. Although acidic oligosaccharides amount to only 14% of the total oligosaccharides, their sialic acid residues occur exclusively as the Sia alpha 2----3Gal group. Removal of the sugar moiety from intact recombinant human interleukin 5 produced a 2.5-fold increase of its activity to induce IgM secretion.     

The third chains of living organisms--a trail of glycobiology that started from the third floor of building 4 in NIH

Application of a finger-printing method to the analysis of human milk oligosaccharides led to the finding that several oligosaccharides were missing in the milk of non-secretor or Lewis-negative individuals. This finding helped us in opening the door of elucidating the enzymatic basis of blood types in human. Based on these successful studies, a strategy to establish reliable techniques to elucidate the structures and functions of the N-linked sugar chains of glycoproteins was devised. It was to contrive enzymatic and chemical means to release quantitatively the N-linked sugar chains as oligosaccharides, and finger-print them by using appropriate methods to demonstrate the sugar pattern of a glycoprotein. These methods enabled us to determine that the N-linked sugar chains of glycoproteins can be classified into three subgroups: high mannose-type, complex-type, and hybrid-type. By comparative studies of the sugar patterns of a glycoprotein produced by different organs and different animals, occurrences of organ- and species-specific glycosylation were found in many glycoproteins. By comparative studies of the glycosylation patterns of the subunits constructing human chorionic gonadotropin and other glycoproteins, occurrence of site-directed N-glycosylation was also found, indicating that the processing and maturation of the N-linked sugar chains of a glycoprotein might be controlled by the structure of polypeptide moiety. Furthermore, these methods enabled us to elucidate the structural alteration of the sugar chains of a glycoprotein induced by diseased state of the producing cells, such as rheumatoid arthritis and malignancy. Recent studies of glycoproteins in the brain-nervous system through aging revealed that N-glycosylation of P(0) in the rat spinal cord is induced by aging. Therefore, glycobiology is expanding tremendously into fields such as pathological and gerontological research.     

On the evolutionary origin of aging

It is generally believed that the first organisms did not age, and that aging thus evolved at some point in the history of life. When and why this transition occurred is a fundamental question in evolutionary biology. Recent reports of aging in bacteria suggest that aging predates the emergence of eukaryotes and originated in simple unicellular organisms. Here we use simple models to study why such organisms would evolve aging. These models show that the differentiation between an aging parent and a rejuvenated offspring readily evolves as a strategy to cope with damage that accumulates due to vital activities. We use measurements of the age-specific performance of individual bacteria to test the assumptions of the model, and find evidence that they are fulfilled. The mechanism that leads to aging is expected to operate in a wide range of organisms, suggesting that aging evolved early and repeatedly in the history of life. Aging might thus be a more fundamental aspect of cellular organisms than assumed so far.     

Resveratrol and rapamycin: are they anti‐aging drugs?

Studies of the basic biology of aging have advanced to the point where anti‐aging interventions, identified from experiments in model organisms, are beginning to be tested in people. Resveratrol and rapamycin, two compounds that target conserved longevity pathways and may mimic some aspects of dietary restriction, represent the first such interventions. Both compounds have been reported to slow aging in yeast and invertebrate species, and rapamycin has also recently been found to increase life span in rodents. In addition, both compounds also show impressive effects in rodent models of age‐associated diseases. Clinical trials are underway to assess whether resveratrol is useful as an anti‐cancer treatment, and rapamycin is already approved for use in human patients. Compounds such as these, identified from longevity studies in model organisms, hold great promise as therapies to target multiple age‐related diseases by modulating the molecular causes of aging.     

Environmental effects on the expression of life span and aging: an extreme contrast between wild and captive cohorts of Telostylinus angusticollis (Diptera: Neriidae)

Most research on life span and aging has been based on captive populations of short-lived animals; however, we know very little about the expression of these traits in wild populations of such organisms. Because life span and aging are major components of fitness, the extent to which the results of many evolutionary studies in the laboratory can be generalized to natural settings depends on the degree to which the expression of life span and aging differ in natural environments versus laboratory environments and whether such environmental effects interact with phenotypic variation. We investigated life span and aging in Telostylinus angusticollis in the wild while simultaneously estimating these parameters under a range of conditions in a laboratory stock that was recently established from the same wild population. We found that males live less than one-fifth as long and age at least twice as rapidly in the wild as do their captive counterparts. In contrast, we found no evidence of aging in wild females. These striking sex-specific differences between captive and wild flies support the emerging view that environment exerts a profound influence on the expression of life span and aging. These findings have important implications for evolutionary gerontology and, more generally, for the interpretation of fitness estimates in captive populations.     

A call to fins! Zebrafish as a gerontological model

Among the wide variety of model organisms commonly used for studies on aging, such as worms, flies and rodents, a wide research gap exists between the invertebrate and vertebrate model systems. In developmental biology, a similar gap has been filled by the zebrafish (Danio rerio). We propose that the zebrafish is uniquely suited to serve as a bridge model for gerontology. With high fecundity and economical husbandry requirements, large populations of zebrafish may be generated quickly and cheaply, facilitating large-scale approaches including demographic studies and mutagenesis screens. A variety of mutants identified in such screens have led to modelling of human disease, including cardiac disorders and cancer. While zebrafish longevity is at least 50% longer than in commonly used mouse strains, as an ectothermic fish species, its life span may be readily modulated by caloric intake, ambient temperature and reproductive activity. These features, coupled with a growing abundance of biological resources, including an ongoing genome sequencing project, make the zebrafish a compelling model organism for studies on aging.     

Recent advances in glycotechnology for glycoconjugate synthesis using microbial endoglycosidases

Biotechnology associated with synthesis of glycopeptides and glycoproteins has recently advanced as glycotechnology. Studies toward glycotechonology include the artificial modification of sugar chains in glycoconjugates to improve their function because the physiological importance of sugar chains in living organisms is well recognized. Methods involving addition of oligosaccharides to peptides and proteins have attracted attention as efficient techniques in glycotechnology, especially those involving the transglycosylation activities of microbial endoglycosidases. The exploration of oligosaccharide oxazolines as donor substrates for the transglycosylation of endoglycosidases has significantly enhanced the efficiency of these processes. Moreover, discovery of novel endoglycosidase mutants with glycosynthase-like activity has made it possible to effectively synthesize large quantities of glycopeptides, as well as homogeneous glycoprotein. The use of mutant enzymes and oligosaccharide oxazolines has led to development of practical applications for the synthesis of bioactive glycopeptides and therapeutic glycoproteins as bio-medicines.     

Functional analyses of human DNA repair proteins important for aging and genomic stability using yeast genetics

Model systems have been extremely useful for studying various theories of aging. Studies of yeast have been particularly helpful to explore the molecular mechanisms and pathways that affect aging at the cellular level in the simple eukaryote. Although genetic analysis has been useful to interrogate the aging process, there has been both interest and debate over how functionally conserved the mechanisms of aging are between yeast and higher eukaryotes, especially mammalian cells. One area of interest has been the importance of genomic stability for age-related processes, and the potential conservation of proteins and pathways between yeast and human. Translational genetics have been employed to examine the functional roles of mammalian proteins using yeast as a pliable model system. In the current review recent advancements made in this area are discussed, highlighting work which shows that the cellular functions of human proteins in DNA repair and maintenance of genomic stability can be elucidated by genetic rescue experiments performed in yeast.