Sialic acid activation.

Cytidine 5'-monophosphosialic acid (CMP-sialic acid) is the activated form of sialic acid which is required for the biosynthesis of sialic acid-containing complex carbohydrates. Its discovery over 30 years ago by the laboratory of Dr Saul Roseman was a landmark in research dealing with the biosynthesis of these compounds. A review is presented of the salient features concerning this molecule: its discovery, chemistry, biosynthesis, subcellular location, enzymatic cleavage, transport and molecular biology. This report does not deal with its utilization by the sialyltransferases.     

At the Revolution with Fred Sherman

Fred Sherman was a prominent yeast geneticist and my mentor in graduate school. Fred passed away in September 2013 at the age of 81. In this minireview, I describe what it was like to know Fred and be in his lab from 1977 to 1982, the extraordinarily exciting time when the recombinant DNA revolution hit yeast genetics.     

August Kappel (1840–1915)

I would like to express my thanks to all those who have helped me in the preparation of this article, in particular my mother, Mrs K. Brett, for supplying information about the Kirby family; my cousin, Johanna Meyer, for information about the Kappel family; my son, Geoffrey Dommett, for his computer expertise; and Gina Douglas, librarian, for making available to me the archives of the Linnean Society.     

Solubilization and enrichment of boar sperm membrane proteins

Boar sperm membranes are rather resistent to the solubilizing effect of some detergents. Deoxycholate, an ionic detergent, was efficient in solubilizing sperm proteins but some nonionic detergents like Triton X-100 displayed relatively poor capacity in rendering membrane proteins soluble. This may be due to sperm proteins being attached to submembraneous structures through bonds involving divalent cations, since mixtures of Triton X-100 and ethylenediamine tetraacetic acid (EDTA) were almost as efficient as deoxycholate in solubilizing membrane proteins. Since intact spermatozoa were directly treated with detergents the solubilized proteins comprised a mixture of intracellular and membrane components. To enrich for membrane proteins, affinity chromatography on columns containing different lectins was carried out. SDS polyacryiamide gel electrophoresis of sperm glycoproteins desorbed from the various lectin columns demonstrated that each lectin bound a unique set of components although most glycoproteins were recovered from two or more columns. Columns containing Lens culinaris hemagglutinin yielded more sperm glycoproteins than any of the other lectin columns examined. The predominant amount of the sperm proteins recovered from the Lens culinaris lectin column was membrane derived, as the majority of the proteins were integrated into liposomes. It is concluded that sperm membrane proteins are efficiently solubilized by detergent in the presence of a chelator and that most of the membrane glycoproteins can easily be enriched by affinity chromatography on a lectin column. Proteins obtained in this way should serve as excellent starting material for the isolation of individual sperm membrane proteins.     

Hepatitis C virus structural proteins and virus-like particles produced in recombinant baculovirus-infected insect cells

Three proteins, namely, the core protein C and envelope glycoproteins E1 and E2, are main structural proteins forming a hepatitis C virus (HCV) virion. The virus structure and assembly and the role of the structural proteins in virion morphogenesis remain unknown because of the lack of an efficient culture system for HCV to be grown in vitro. Highly efficient heterologous expression systems make it possible to obtain self-assembled, nonreplicating, genome-lacking particles that are morphologically similar to intact virions. Using recombinant baculoviruses expressing the HCV structural protein genes in insect cells, the individual HCV structural proteins were expressed to 25–35% of the total cell protein, and the CE1 and E1E2 heterodimers and HCV-like particles were obtained. It was demonstrated that the recombinant C, E1, and E2 proteins underwent posttranslational modification, the glycoproteins formed a noncovalent heterodimer, and HCV- like particles were located in endoplasmic reticulum membranes of infected cells. The formation of E1E2 dimers and HCV-like particles was used to study the effect of E1 glycosylation on the expression and processing of the coat proteins.     

Two-dimensional electrophoretic analysis of the regulation of SOS proteins in three ssb mutants

A previously undescribed mutation in the ssb gene, which codes for a major single strand DNA binding protein essential for DNA repelication, was mapped on the Escherichia coli Chromosome. Three ssb mutants were analyzed under parallel physiological conditions for the induction of SOS proteins (products of recA, uvrA, and an unknown gene), the production of mutants, the induction of lambda prophage, and sensitivity to DNA damaging agents. Two-dimensional electrophoretic techniques were used to quantitate changes in the rate of synthesis of proteins. The previously unpublished position of the uvrA gene-product in the two-dimensional matrix of E. coli proteins was described. These ssb strains exhibited varying sensitivities to ultraviolet irradiation and methylmethane sulfonate that correlated with the rate of constitutive synthesis of SOS proteins, spontaneous commitment to virulent growth of lambda lysogens, and elevation of endogenous mutation rates.Dedicated to the memory of Roger Y. Stanier: to his fascination for diverse microbial lifeforms that catalyzed curiosity in his associates, to his intellectual aura that elicited deep respect, to his pursuit of scientific truth that promoted the highest research ethics, to his friendly nature that encouraged my growth as a scientist and enkindled my love for Roger     

A Reminiscence

Leslie Orgel and Francis Crick with Gobind Khorana in Madison, Wisconsin (December 1965). I first met Leslie at the Endicott House (MIT) in February 1964. Leslie was then spending a period of time at MIT and the occasion was a party for him. During our conversation, Leslie talked about starting some experimental work. He seemed to be particularly interested in polyphosphates and the chemical activation of small molecules (building blocks).Shortly after his move to the Salk Institute in the Fall of 1964 I visited him in January 1965. He already had a lab going. I remember meeting Jim Ferris, in particular, and John Sulston sometime later. That particular time was exciting for my research as well. We had the first results on the Genetic Code using the chemical-biochemical approach that my lab had developed. Francis Crick was also at the Salk Institute during the time of my visit. Both Leslie and Francis were very excited by my results and they began to ask a lot of questions and gave me a whole lot of suggestions about further experiments. In fact, my thinking and planning of things that we were doing were so scrutinized and clarified during these discussions that, it seemed to me, my own group had only to turn out all the experiments that were needed. These interactions with Francis and Leslie continued intensively throughout that year and later. In fact, both Leslie and Francis accepted my invitation to Madison in December 1965 for more discussions.Since those early days of the Salk Institute, I have made numerous visits over the years to Leslie and his research group. It has always been very exciting to learn about the many discoveries bearing on chemical evolution that have unfolded from Leslie's research group. In addition, I have always benefitted from the insightful comments that Leslie invariably provided on my own research. I look forward to our continued interactions and friendship in the future.Leslie, A Happy Birthday!     

The paradigm of Huntington disease.

I recall it as vividly as though it had occurred but yesterday. It made a most enduring impression upon my boyish mind which was my very first impulse to choosing chorea as my virgin contribution to medical lore. Driving with my father through a wooded road leading from East Hampton to Amagansett we suddenly came upon two women, mother and daughter, both tall and thin, almost cadaverous, both bowing, twisting, grimacing. I stared in wonderment, almost in fear. What could it mean? My father paused to speak with them and we passed on. Then my Gamaliel-like instruction began; my medical education had its inception. From this point on, my interest in the disease has never wholly ceased. [George Huntington, at 59, recalling how at the age of 8 years he first saw Huntington disease while traveling with his physician father on his professional rounds in 1858].     

Novel eicosanoid pathways

This article reviews a lecture I was honored to present at the Leon Wolfe Symposium in Montreal on March 25, 2004. The lecture described my research career, which started with my interaction with Wolfe at the Montreal Neurological Institute as a postdoctoral fellow and research associate and was followed by additional research discoveries after I left Montreal for my first academic position at the Research Institute, The Hospital for Sick Children and University of Toronto. The article consists of two parts. The first part involves the discovery (in Wolfe’s laboratory) of a new pathway of arachidonic acid, in which a bicyclic prostanoid structure (later called prostacyclin by John Vane and his group) was described, and its further development in Toronto, which led to the discovery of the conversion of the bicyclic prostanoid into 6-keto prostaglandin F_1α. The second part deals with the hepoxilin pathway, a pathway I discovered during a sabbatical leave in Japan with Professor Shozo Yamamoto, which was followed by a stay of several months in the laboratory of Professor Bengt Samuelsson in Sweden. I deal with the historical aspects of both pathways and end with interesting novel aspects of hepoxilin stable antagonist analogs in the treatment of solid tumors in experimental animals.     

水稻可溶性糖蛋白的纯化与鉴定研究

从水稻鲜叶中提取总蛋白,对总蛋白中的蛋白质含量进行了测定;通过硫酸铵沉淀将总蛋白提取液进行分级,从而达到了总蛋白细分和放量的目的。四级份的分级蛋白分别通过ConA-Sepharose 4B 亲和层析进行糖蛋白纯化,按吸收峰收集的各级糖蛋白混合物进行冷冻干燥,得到干粉;结合PAS法染色和考马斯亮蓝R-250染色对四级份的糖蛋白样品鉴定,在其中3个级份中均检测出糖蛋白;由于感度的差异,按考马斯亮蓝R-250染色可检测出近30种糖蛋白(包括部分糖肽),按PAS法染色可检测到7种糖蛋白;对3种含量较高的糖蛋白进行了胶上纯化,3种糖蛋白的PAS法染色均证实了3种样品为单一的糖蛋白或者糖肽,分别命名为RG1、RG2和RG3。     

Intracellular trafficking of Gag and Env proteins and their interactions modulate pseudotyping of retroviruses

Glycoproteins derived from most retroviruses and from several families of enveloped viruses can form infectious pseudotypes with murine leukemia virus (MLV) and lentiviral core particles, like the MLV envelope glycoproteins (Env) that are incorporated on either virus type. However, coexpression of a given glycoprotein with heterologous core proteins does not always give rise to highly infectious viral particles, and restrictions on pseudotype formation have been reported. To understand the mechanisms that control the recruitment of viral surface glycoproteins on lentiviral and retroviral cores, we exploited the fact that the feline endogenous retrovirus RD114 glycoprotein does not efficiently pseudotype lentiviral cores derived from simian immunodeficiency virus, whereas it is readily incorporated onto MLV particles. Our results indicate that recruitment of glycoproteins by the MLV and lentiviral core proteins occurs in intracellular compartments and not at the cell surface. We found that Env and core protein colocalization in intracytoplasmic vesicles is required for pseudotype formation. By investigating MLV/RD114 Env chimeras, we show that signals in the cytoplasmic tail of either glycoprotein differentially influenced their intracellular localization; that of MLV allows endosomal localization and hence recruitment by both lentiviral and MLV cores. Furthermore, we found that upon membrane binding, MLV core proteins could relocalize Env glycoproteins in late endosomes and allow their incorporation on viral particles. Thus, intracellular colocalization, as well as interactions between Env and core proteins, may influence the recruitment of the glycoprotein onto viral particles and generate infectious pseudotyped viruses.     

Glycoproteomic profile in wine: a 'sweet' molecular renaissance

Glycoproteins are believed to be important in several technological, oenological and allergological processes due to their physicochemical properties. The knowledge of the protein glycosylation status in wine will aid in the understanding of these processes. A multiplexed glycopeptide enrichment strategy in combination with tandem mass spectrometry was performed in order to analyze the glycoproteome of white wine. A total of 28 glycoproteins and 44 glycosylation sites were identified. The identified glycoproteins were from grape and yeast origin. In particular, several glycoproteins derived from grape, like invertase and pathogenesis-related (PR) proteins, and from the yeast, were found after the vinification process. Bioinformatic analysis revealed sequence similarity between the identified grape glycoproteins and known plant allergens. This study is an important step forward in order to investigate the implication of glycoproteins in several processes, like protein stabilization and potential allergenic cross-reactivity in wine.     

Sampling phylogenetic tree space with the generalized Gibbs sampler

A recent article published in Cladistics is critical of a number of heuristic methods for phylogenetic inference based on parsimony scores. One of my papers is among those criticized, and I would appreciate the opportunity to make a public response. The specific criticism is that I have re‐invented an algorithm for economizing parsimony calculations on trees that differ by a subtree pruning and regrafting (SPR) rearrangement. This criticism is justified, and I apologize for incorrectly claiming originality for my presentation of this algorithm. However, I would like to clarify the intent of my paper, if I can do so without detracting from the sincerity of my apology. My paper is not about that algorithm, nor even primarily about parsimony. Rather, it is about a novel strategy for Markov chain Monte Carlo (MCMC) sampling in a state space consisting of trees. The sampler involves drawing from conditional distributions over sets of trees: a Gibbs‐like strategy that had not previously been used to sample tree‐space. I would like to see this technique incorporated into MCMC samplers for phylogenetics, as it may have advantages over commonly used Metropolis‐like strategies. I have recently used it to sample phylogenies of a biological invasion, and I am finding many applications for it in agent‐based Bayesian ecological modelling. It is thus my contention that my 2005 paper retains substantial value.     

Ape-like endocast of “ape-man” Taung

I have identified and illustrated a spherical “dimple” or “depression” on the Taung endocast as indicating the most likely position of the medial end of the lunate sulcus but have not drawn an actual lunate sulcus on Taung because one is not visible. In a recent paper, R.L. Holloway (Am. J. Phys. Anthropol. 77:27–33, 1988) drew a lunate sulcus on his copy of the Taung endocast, incorrectly attributed this sulcus to me, and used it to obtain a ratio of 0.254 to describe “Falk's” position of the lunate sulcus. My published ratio of 0.242 for Taung (Falk: Am. J. Phys. Anthropol. 67:313–315, 1985a) was not considered, although the focus of Holloway's paper was my assessment of the position of the lunate sulcus. Holloway also excluded published ratios for a chimpanzee in my collection from his statistical analysis but, even so, my published ratio for Taung is still only 1.5 standard deviations from his chimpanzee mean. If my chimpanzee brain is included in the sample, the ratio for Taung is 1.2 standard deviations from the mean. Furthermore, one of Holloway's own chimpanzees (B60–7) has a ratio of 0.241, just 0.001 below my ratio for Taung. There is no sulcus where Holloway has drawn one on Taung, his “F(LS)” is not mine, his 2 mm error is not mine, and the correct ratio for my measurement of Tuang is the one that I published, not the one that Holloway attributes to me. Assessment of Holloway's chimpanzee data supports my claim that the dimple on the Taung endocast is within the chimpanzee range for the medial end of the lunate sulcus.     

The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells

Orally ingested botulinum toxin enters the circulatory system and eventually reaches the peripheral nerves, where it elicits a response of neurological dysfunction. In this study, we report the important findings concerning the mechanism of Clostridium botulinum type C progenitor toxin (C16S) endocytic mechanism. C16S toxin bound to high molecular weight proteins on the surface of human colon carcinoma HT-29 cells and was internalized, but not if the cells were pretreated with neuraminidase. Benzyl-GalNAc which inhibited O-glycosylation of glycoproteins also interfered in the toxin's ability to bind the cell surface. On the other hand, the toxin was internalized in spite of pretreatment of the cells with PPMP, an inhibitor of ganglioside synthesis. These results suggest that the glycoproteins, like mucin, fulfill the important roles of receptor and transporter of C16S toxin.     

A moment at the cell centre

I have been invited by the board of the French Society of Cell Biology (SBCF) to write a text around my presentation in the Symposium ‘A day at the Cell Centre’, held at the Curie Institute on May 17, 2019, and organized by four of my former students, namely Juliette Azimzadeh, Nathalie Delgehyr, Matthieu Piel and Manuel Théry. I have to thank them warmly for the quality of the science during this day. It was also a moving day for me indeed to listen to so many figures in the field.     

Molecular cloning and heterologous expression of beta1,2-xylosyltransferase and core alpha1,3-fucosyltransferase from maize

Maize is considered a promising alternative production system for pharmaceutically relevant proteins. However, like in all other plant species asparagine-linked oligosaccharides of maize glycoproteins are modified with beta1,2-xylose and core alpha1,3-fucose sugar residues, which are considered to be immunogenic in mammals. This altered N-glycosylation when compared to mammalian cells may reduce the potential of maize as a production system for heterologous glycoproteins. Here we report the cloning and characterization of the cDNA sequences coding for the maize enzymes beta1,2-xylosyltransferase (XylT) and core alpha1,3-fucosyltransferase (FucT). The cloned XylT and FucT cDNAs were shown to encode enzymatically active proteins, which were independently able to convert a mammalian acceptor glycoprotein into an antigen binding anti-plant N-glycan antibodies. The complete sequence of the XylT gene was determined. Evidence for the presence of at least three XylT and FucT gene loci in the maize genome was obtained. The identification of the two enzymes and their genes will allow the targeted downregulation or even elimination of beta1,2-xylose and core alpha1,3-fucose addition to recombinant glycoproteins produced in maize.     

Bacterial glycoproteins: functions,biosynthesis and applications

Although widely distributed in eukaryotic cells glycoproteins appear to be rare in prokaryotic organisms. The prevalence of the misconception that bacteria do not glycosylate their proteins has been a subject matter of discussion for a long time. Glycoconjugates that are linked to proteins or peptides, generated by the ribosomal translational mechanism have been reported only in the last two to three decades in a few prokaryotic organisms. Most studied prokaryotic glycoproteins are the S-layer glycoproteins of Archeabacteria. Apart from these, membrane-associated, surface-associated, secreted glycoproteins and exoenzymes glycoproteins are also well documented in both, Archea and Eubacteria. From the recent literature, it is now clear that prokaryotes are capable of glycosylating proteins. In general, prokaryotes are deprived of the cellular organelles required for glycosylation. In prokaryotes many different glycoprotein structures have been observed that display much more variation than that observed in eukaryotes. Besides following similar mechanisms in the process of glycosylation, prokaryotes have also been shown to use mechanisms that are different from those found in eukaryotes. The knowledge pertaining to the functional aspects of prokaryotic glycoproteins is rather scarce. This review summarizes developments and understanding relating to characteristics, synthesis, and functions of prokaryotic glycoproteins. An extensive summary of glycosylation that has been reported to occur in bacteria has also been tabulated. Various possible applications of these diverse biomolecules in biotechnology, vaccine development, pharmaceutics and diagnostics are also touched upon.