Chemoenzymatic synthesis of 2-azidoethyl-ganglio-oligosaccharides GD3, GT3, GM2, GD2, GT2, GM1, and GD1a

We have synthesized several ganglio-oligosaccharide structures using glycosyltransferases from Campylobacter jejuni. The enzymes, alpha-(2-->3/8)-sialyltransferase (Cst-II), beta-(1-->4)-N-acetylgalactosaminyltransferase (CgtA), and beta-(1-->3)-galactosyltransferase (CgtB), were produced in large-scale fermentation from Escherichia coli and further characterized based on their acceptor specificities. 2-Azidoethyl-glycosides corresponding to the oligosaccharides of GD3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT3 (alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-), GM2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GD2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), GT2 (beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->8)-alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-), and GM1 (beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) were synthesized in high yields (gram-scale). In addition, a mammalian alpha-(2-->3)-sialyltransferase (ST3Gal I) was used to sialylate GM1 and generate GD1a (alpha-D-Neup5Ac-(2-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1-->4)-[alpha-D-Neup5Ac-(2-->3)]-beta-D-Galp-(1-->4)-beta-D-Glcp-) oligosaccharide. We also cloned and expressed a rat UDP-N-acetylglucosamine-4'epimerase (GalNAcE) in E. coli AD202 cells for cost saving in situ conversion of less expensive UDP-GlcNAc to UDP-GalNAc.     

Chromosome localization of the loci for PEPA,PEPB, PEPS, 1DH1, GSR,MPI, PGM1, NP,SOD1, and ME1 in the common shrew (Sorex araneus)

This report extends the genetic map of the common shrew (Sorex araneus) by adding chromosome assignments for ten genes to the seven already mapped (Pack et al. 1995). A somatic cell hybrid panel was used for the mapping. The genes for peptidase A (PEPA) and isocitrate dehydrogenase-1 (IDH1) map to chromosome de; the genes for phosphoglucomutase-1 (PGM1), superoxide dismutase-1 (SOD1), and mannosephosphate isomerase (MPI) are located on chromosome af; the genes for nucleoside phosphorylase (NP) and glutathione reductase (GSR) are on chromosome ik; and the genes for peptidase S (PEPS), malic enzyme-1 (ME1), peptidase B (PEPB) are found on chromosomes jl, go, and mp respectively. Received: 2 October 1995 / Accepted: 21 November 1995     

The Effect of Mustard Gas on Salivary Trace Metals (Zn,Mn, Cu,Mg, Mo,Sr, Cd,Ca, Pb,Rb)

We have determined and compared trace metals concentration in saliva taken from chemical warfare injures who were under the exposure of mustard gas and healthy subjects by means of inductively coupled plasma optical emission spectroscopy (ICP-OES) for the first time. The influence of preliminary operations on the accuracy of ICP-OES analysis, blood contamination, the number of restored teeth in the mouth, salivary flow rate, and daily variations in trace metals concentration in saliva were also considered. Unstimulated saliva was collected at 10:00–11:00 a.m. from 45 subjects in three equal groups. The first group was composed of 15 healthy subjects (group 1); the second group consisted of 15 subjects who, upon chemical warfare injuries, did not use Salbutamol spray, which they would have normally used on a regular basis (group 2); and the third group contained the same number of patients as the second group, but they had taken their regular medicine (Salbutamol spray; group 3). Our results showed that the concentration of Cu in saliva was significantly increased in the chemical warfare injures compared to healthy subjects, as follows: healthy subjects 15.3± 5.45(p.p.b.), patients (group 2) 45.77±13.65, and patients (Salbutamol spray; group 3) 29 ±8.51 (P <0.02). In contrast, zinc was significantly decreased in the patients, as follows: healthy subjects 37 ± 9.03(p.p.b.), patients (group 2) 12.2 ± 3.56, and patients (Salbutamol spray; group 3) 20.6 ±10.01 (P < 0.01). It is important to note that direct dilution of saliva samples with ultrapure nitric acid showed the optimum ICP-OES outputs.     

Aerobic Biotransformation of N-Nitrosodimethylamine and N-Nitrodimethylamine by Benzene-, Butane-, Methane-, Propane-, and Toluene-Fed Cultures

N-Nitrosodimethylamine (NDMA) is an emerging contaminant of concern. N-nitrodimethylamine (DMNA) is a structural analog to NDMA. NDMA and DMNA have been found in drinking water, groundwater, and other media and are of concern due their toxicity. The authors evaluated biotransformation of NDMA and DMNA by cultures enriched from contaminated groundwater growing on benzene, butane, methane, propane, or toluene. Maximum specific growth rates of enriched cultures on butane (μ_max = 1.1 h^?1) and propane (μ_max = 0.65 h^?1) were 1 to 2 orders of magnitude higher than those presented in the literature. Growth rates of mixed cultures grown on benzene (μ_max = 1.3 h^?1), methane (μ_max = 0.09 h^?1), and toluene (μ_max = 0.99 h^?1) in these studies were similar to those presented in the literature. NDMA biotransformation rates for methane oxidizers (υ_max = 1.4 ng min^?1 mg^?1) and toluene oxidizers (υ_max = 2.3 ng min^?1 mg^?1) were comparable to those presented in the literature, whereas the biotransformation rate for propane oxidizers (υ_max = 0.37 ng min^?1 mg^?1) was lower. NDMA biotransformation rates for benzene oxidizers (υ_max = 1.02 ng min^?1 mg^?1) and butane oxidizers (υ_max = 1.2 ng min^?1 mg^?1) were comparable to those reported for other primary substrates. These studies showed that DMNA biotransformation rates for benzene (υ_max = 0.79 ng min^?1 mg^?1), butane (υ_max = 1.0 ng min^?1 mg^?1), methane (υ_max = 2.1 ng min^?1 mg^?1), propane (υ_max = 1.46 ng min^?1 mg^?1), and toluene (υ_max = 0.52 ng min^?1 mg^?1) oxidizers were all comparable. These studies highlight potential bioremediation methods for NDMA and DMNA in contaminated groundwater.     

Synthesis,tautomerism, and antimicrobial,anti-HCV,anti-SSPE,antioxidant, and antitumor activities of arylazobenzosuberones

2-Dimethylaminomethylene-1-benzosuberone 1 was coupled with diazotized aniline derivatives to afford a series of the hitherto unreported 2-arylazo-1-benzosuberones 3a–i. The tautomeric structure and the effect of substituents on the tautomeric form (s) of the products 3a–i were discussed. Similar coupling of the enaminone 1 with diazonium salts of heterocyclic amines gave the respective fused azolotriazino-benzosuberones. Some of the newly synthesized compounds showed potent antimicrobial, anti-HCV, antioxidant, antitumor (as topoisomerase I inhibitors), and antimicrobial activities.     

Synthesis,Chemical Characterization,DNA Binding,Antioxidant, Antibacterial,and Antifungal Activities of Ferrocence Incorporated Selenoureas

Ferrocene‐incorporated selenoureas 1‐(4‐methoxybenzoyl)‐3‐(4‐ferrocenylphenyl)selenourea (P4Me), 1‐(3‐methoxybenzoyl)‐3‐(4‐ferrocenylphenyl)selenourea (P3Me), and 1‐(2‐methoxybenzoyl)‐3‐(4‐ferrocenylphenyl)selenourea (P2Me) were synthesized and characterized by nuclear magnetic resonance, Fourier transform infrared spectroscopy, atomic absorption spectroscopy, CHNS, and single‐crystal X‐ray diffraction. DNA interaction of the compounds was investigated with cyclic voltammetry, UV–visible spectroscopy, and viscometry, which is a prerequisite for anticancer agents. Drug‐DNA binding constant was found to vary in the sequence: K_P4Me (4.9000 × 10⁴ M^?1) > K_P2Me (2.318 × 10⁴ M^?1) > K_P3Me (1.296 × 10⁴ M^?1). Antioxidant (1,1‐diphenyl‐2‐picrylhydrazyl), antifungal (against Faussarium solani and Helmentosporium sativum), and antibacterial (against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus subtilis) activities have also been reported in addition.     

A facile and effective synthesis of alpha-(1-->6)-linked mannose di-, tri-, tetra-, hexa-, octa-, and dodecasaccharides, and beta-(1-->6)-linked glucose di-, tri-, tetra-, hexa-, and octasaccharides using sugar trichloroacetimidates as the donors and unprotected or partially protected glycosides as the acceptors

Reaction of 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl trichloroimidate with allyl alpha-D-mannopyranoside in the presence of TMSOTf selectively gave allyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranoside through an orthoester intermediate. Benzoylation of 3, followed by deallylation, and then trichloroimidation afforded the disaccharide donor 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroimidate, while benzoylation of 3 followed by selective removal of acetyl groups yielded the disaccharide acceptor allyl alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside. Coupling of 5 with 6 gave the tetrasaccharide allyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside, which were converted into the tetrasaccharide donor 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl trichloroimdate and the tetrasaccharide acceptor allyl alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzoyl-alpha-D-mannopyranoside, respectively, by the same strategies as used for conversion of 3 into 5 and 6. Condensation of 5 with 13 gave the hexasaccharide 14, while condensation of 12 with 13 gave the octasaccharide 17. Dodecasaccharide 21 was obtained by the coupling of 12 with the octasaccharide acceptor 20. Similar strategies were used for the syntheses of beta-(1-->6)-linked glucose di-, tri-, tetra-, hexa-, and octamers. Deprotection of the oligosaccharides in ammonia-saturated methanol yielded the free alpha-(1-->6)-linked mannosyl and beta-(1-->6)-linked glucosyl oligomers.     

Biotransformation of isoquinoline, phenanthridine, phthalazine, quinazoline, and quinoxaline by Streptomyces viridosporus

Streptomyces viridosporus T7A (ATCC␣39115), during growth in tryptone/yeast extract broth, cometabolized five heterocyclic nitrogen-containing compounds. The metabolites produced from the azaarenes were identified by high-performance liquid chromatography, UV/visible absorption spectroscopy, and mass spectrometry. Isoquinoline was transformed to 1(2H)-isoquinolinone (14%), phenanthridine to 6(5H)-phenanthridinone (25%), phthalazine to 1(2H)-phthalazinone (46%), quinazoline to 2,4(1H,3H)-quinazolinedione (4%), and quinoxaline to 2(1H)-quinoxalinone (8%) and 1-methyl-2(1H)-quinoxalinone (12%). Received: 29 July 1997 / Received revision: 19 November 1997 / Accepted: 29 November 1997     

Penta-, hexa-, and heptasaccharide acceptor products of alternansucrase

In the presence of suitable acceptor molecules, dextransucrase makes a homologous series of oligosaccharides in which the isomers differ by a single glucosyl unit, whereas alternansucrase synthesizes one trisaccharide, two tetrasaccharides, etc. For the example of maltose as the acceptor, if one considers only the linear, unbranched possibilities for alternansucrase, the hypothetical number of potential products increases exponentially as a function of the degree of polymerization (DP). Experimental evidence indicates that far fewer products are actually formed. We show that only certain isomers of DP >4 are formed from maltose in measurable amounts, and that these oligosaccharides belong to the oligoalternan series rather than the oligodextran series. When the oligosaccharide acceptor products from maltose were separated by size-exclusion chromatography and HPLC, only one pentasaccharide was isolated. Its structure was alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Two hexasaccharides were formed in approximately equal quantities: alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc and alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. Just one heptasaccharide was isolated from the reaction mixture, alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->3)-alpha-D-Glcp-(1-->6)-alpha-D-Glcp-(1-->4)-D-Glc. We conclude that the enzyme is incapable of forming two consecutive alpha-(1-->3) linkages, and does not form products with more than two consecutive alpha-(1-->6) linkages. The distribution of products may be kinetically determined.     

Synthesis,Characterization, Cellular Uptake,Apoptosis, Cytotoxicity,Dna-Binding,and Antioxidant Activity Studies of Ruthenium(II) Complexes

Two new ruthenium(II) polypyridyl complexes [Ru(dmb)₂(HECIP)](ClO₄)₂ (1) (HECIP = N-ethyl-4-[(1,10)-phenanthroline(5,6-f)imidazol-2-yl]carbazole, dmb = 4,4’-dimethyl-2,2’-bipyridine) and [Ru(dmp)₂(HECIP)](ClO₄)₂ (2) (dmp = 2,9-dimethyl-1,10-phenanthroline) have been synthesized and characterized. The DNA-binding behaviors of the two complexes were investigated by absorption spectra, viscosity measurements, and photoactivated cleavage. The DNA-binding constants for complexes 1 and 2 were determined to be 8.03 (± 0.12) × 10⁴ M^?1 (s = 1.62) and 2.97 (± 0.15) × 10⁴ M^?1 (s = 1.82), respectively. The results suggest that these complexes interact with DNA through intercalative mode. The photocleavage of pBR322 DNA by Ru(II) complexes was investigated. The cytotoxicity of complexes 1 and 2 has been evaluated by the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)] method. Complex 1 shows higher anticancer potency than 2 against the four tumor cell lines. Apoptosis and cellular uptake were investigated. The antioxidant activities of the ligand and these complexes were also performed.     

Cover Image,Volume 120, Number 11, November 2019

Skeletal tissue homeostasis is maintained via the balance of osteoclastic bone resorption and osteoblastic bone formation. Autophagy and apoptosis are essential for the maintenance of homeostasis and normal development in cells and tissues. We found that Bax-interacting factor 1 (Bif-1/Endophillin B1/SH3GLB1), involving in autophagy and apoptosis, was upregulated during osteoclastogenesis. Furthermore, mature osteoclasts expressed Bif-1 in the cytosol, particularly the perinuclear regions and podosome, suggesting that Bif-1 regulates osteoclastic bone resorption. Bif-1-deficient (Bif-1 ^−/−) mice showed increased trabecular bone volume and trabecular number. Histological analyses indicated that the osteoclast numbers increased in Bif-1 ^−/− mice. Consistent with the in vivo results, osteoclastogenesis induced by receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) was accelerated in Bif-1 ^−/− mice without affecting RANKL-induced activation of RANK downstream signals, such as NF-κB and mitogen-activated protein kinases (MAPKs), CD115/RANK expression in osteoclast precursors, osteoclastic bone-resorbing activity and the survival rate. Unexpectedly, both the bone formation rate and osteoblast surface substantially increased in Bif-1 ^−/− mice. Treatment with β-glycerophosphate (β-GP) and ascorbic acid (A.A) enhanced osteoblastic differentiation and mineralization in Bif-1 ^−/− mice. Finally, bone marrow cells from Bif-1 ^−/− mice showed a significantly higher colony-forming efficacy by the treatment with or without β-GP and A.A than cells from wild-type (WT) mice, suggesting that cells from Bif-1 ^−/− mice had higher clonogenicity and self-renewal activity than those from WT mice. In summary, Bif-1 might regulate bone homeostasis by controlling the differentiation and function of both osteoclasts and osteoblasts (235 words).     

T-cell receptor variable alpha (TCRAV) polymorphisms in European, Chinese, South American, AfroCaribbean, and Gambian populations

 Interactions involving the T-cell receptor (TCR) and major histocompatibility complex (MHC) are fundamental to the generation of a specific immune response. The study of interpopulation differences in TCR genes may identify those genes which are subject to selection, and also provides useful information for future genetic studies in these populations. In this study we present analysis of five TCRAV polymorphisms, for V5S1, V6S1, V8S1, V17S1, and V21S1 loci in five human populations by single-strand conformational polymorphism (SSCP) analysis. Caucasian, Chinese, Gambian, AfroCaribbean, and South American Indians (Mapuches) showed marked interpopulation variation for both the silent (V5S1, V17S1, and V21S1) and coding (V6S1 and V8S1) polymorphisms. In general the alleles were conserved in the different populations, but new, additional variants were found for V5S1 and V17S1 in Gambians and Caucasians. V6S1 overall showed the highest nucleotide diversity, and V6S1 genotype distributions were skewed away from expected values in Chinese and Mapuches. Analysis of allelic associations showed a general lack of linkage disequilibrium between the loci, which was reflected by the absence of strong population-specific haplotypes. Received: 22 April 1997 / Revised: 9 July 1997     

Spirostanol saponins from Paris polyphylla,structures of polyphyllin C,D, E and F

The structures of four new saponins, polyphyllin C, D, E and F, isolated from the tubers of Paris polyphylla have been elucidated as diosgenin-3-O-α-l-rhamnopyranosyl(1→3)-β-d-glucopyranoside, diosgenin-3-O-α-l-rhamnopyranosyl(1→3)- [α-l-arabinofuranosyl(1→4)]-β-d-glucopyranoside, diosgenin-3-O-α-l-rhamnopyranosyl(1→2)-α-l-rhamnopyranosyl (1→4)[α-l-rhamnopyranosyl(1→3)]-β-d-glucopyranoside and diosgenin-3-O-α-l-rhamnopyranosyl(1→4)[α-l- rhamnopyranosyl(1→3)][β-d-glucopyranosyl(1→2)]-α-l-rhamnopyranoside, respectively, on the basis of chemical and spectral data.     

Endocytosis: Past,Present, and Future

Endocytosis may have been a driving force behind the evolution of eukaryotic cells. It plays critical roles in cell biology (e.g., signal transduction) and in organismal physiology (e.g., tissue morphogenesis).Endocytosis, the process of cellular ingestion, may have been the driving force behind evolution of the eucaryotic cell (de Duve 2007). Acquiring the ability to internalize macromolecules and digest them intracellularly would have allowed primordial cells to move out from their food sources and pursue a predatory existence; one that might have led to the development of endosymbiotic relationships with mitochondria and plastids. Thus, it is fitting that endocytosis was first discovered and named as the processes of cell “eating” and “drinking.” In 1883, the developmental biologist Ilya Metchnikoff coined the term phagocytosis, from the Greek “phagos” (to eat) and “cyte” (cell), after observing motile cells in transparent starfish larva surround and engulf small splinters that he had inserted (Tauber 2003). Decades later, in 1931, Warren H. Lewis, one of the earliest cell “cinematographers” coined the term pinocytosis, from the Greek “pinean” (to drink), after observing the uptake of surrounding media into large vesicles in cultured macrophages, sarcoma cells, and fibroblasts by time-lapse imaging (Lewis 1931; Corner 1967).Importantly, these pioneering studies also revealed that the function of endocytosis goes well beyond eating and drinking. Indeed, Metchnikoff, considered one of the founders of modern immunology, realized that the phagocytic behavior of the mesodermal amoeboid cells he had observed under the microscope could serve as a general defense system against invasive parasites, in the larva as in man. This revolutionary concept, termed the phagocytic theory, earned Metchnikoff the 1908 Nobel Prize in Physiology or Medicine for his work on phagocytic immunity, which he shared with Paul Ehrlich who discovered the complementary mechanisms of humoral immunity that led to the identification of antibodies (Vaughan 1965; Tauber 2003; Schmalstieg and Goldman 2008). The phagocytic theory was a milestone in immunology and cell biology, and formally gave birth to the field of endocytosis.Key discoveries over the intervening years, aided in large part by the advent of electron microscopy, revealed multiple pathways for endocytosis in mammalian cells that fulfill multiple critical cellular functions (Fig. 1). These mechanistically and morphologically distinct pathways, and their discoverers, include clathrin-mediated endocytosis (Roth and Porter 1964), caveolae uptake (Palade 1953; Yamada 1955), cholesterol-sensitive clathrin- and caveolae-independent pathways (Moya et al. 1985; Hansen et al. 1991; Lamaze et al. 2001), and, more recently, the large capacity CLIC/GEEC pathway (Kirkham et al. 2005). In place of Metchnikoff’s splinters, many of these discoveries resulted from the detection and tracking of internalized HRP-, ferritin-, or gold-conjugated ligands by electron microscopy. These electron-dense tracers allowed researchers to identify cellular structures associated with the uptake and intracellular sorting of receptor-bound ligands. A particularly striking example is the pioneering work of Roth and Porter, who in 1964 observed the uptake of yolk proteins into mosquito oocytes. To synchronize uptake, they killed female mosquitos at timed intervals after a blood feed and observed the sequential appearance of electron-dense yolk granules in coated pits, coated and uncoated vesicles, and progressively larger vesicles. Their remarkable observations accurately described coated vesicle budding, uncoating, homo- and heterotypic fusion events, as well as the emergence of tubules from early endosomes (Fig. 2), all of which are now known hallmarks of the early endocytic trafficking events.Open in a separate windowFigure 1.Time line for discoveries of endocytic pathways and their discoverers. Boxes are color-coded by pathway. *, Nobel laureate. HRP, horseradish peroxidase; CCVs, clathrin-coated vesicles; CCPs, clathrin-coated pits; EGFR, epidermal growth factor receptor; PM, plasma membrane; ER, endoplasmic reticulum; CLIC/GEEC, clathrin-independent carriers/GPI-enriched endocytic compartments.Open in a separate windowFigure 2.Fiftieth anniversary of the discovery of clathrin-mediated endocytosis by Roth and Porter (1964). The image is the hand-drawn summary of observations made by electron microscopic examination of the trafficking of yolk proteins in a mosquito oocyte. Note the many details, later confirmed and mechanistically studied over the intervening 50 years. These include the growth, invagination, and pinching off of coated pits (1,2), which concentrate cargo taken up by coated vesicles (3), the rapid uncoating of nascent-coated vesicles (4), homotypic fusion of nascent endocytic vesicles in the cell periphery (5), the formation of tubules from early endosomes (7), and changes in the intraluminal properties of larger endosomes (6). Finally, yolk proteins are stored in granules as crystalline bodies (8). (From Roth and Porter 1964; reprinted, with express permission, from Rockefeller University Press © 1964, The Journal of Cell Biology 20: 313–332, doi: 10.1083/jcb.20.2.313.)Another milestone in the field of endocytosis was the discovery of the lysosome by Christian de Duve (Appelmans et al. 1955). Whereas the finding of phagocytosis and other endocytic pathways was possible through microscopy, the discovery of lysosomes originated from a biochemical approach (Courtoy 2007), which benefited from the invention of the ultracentrifuge. de Duve and coworkers observed that preparations of acid phosphatase obtained by subcellular fractionation had an unusual behavior: contrary to most enzymatic activities, the activity of acid phosphatase increased rather than decreased with time, freezing–thawing of the fractions and the presence of detergents. Interestingly, the same was true for other hydrolases, which depended on acidic pH for their optimal activity. This led him to postulate that the acid hydrolases were contained in acidified membrane-bound vesicles. In collaboration with Alex Novikoff, he visualized these vesicles, the lysosomes, by electron microscopy (Beaufay et al. 1956) and later showed their content of acid phosphatase (Farquhar et al. 1972). In 1974, de Duve was awarded the Nobel Prize for Physiology or Medicine for his seminal finding of the lysosomes and peroxisomes. He shared it with Albert Claude and George E. Palade “for their discoveries concerning the structural and functional organization of the cell.” The importance of this work lies also in the significant therapeutic applications that followed. The discovery by Elizabeth Neufeld and collaborators of uptake of lysosomal enzymes by cells provided the foundation for enzyme replacement therapy for lysosomal storage disorders (Neufeld 2011).In the 1970s, research in endocytosis entered the molecular era. Using de Duve and Albert Claude-like methods of subcellular fractionation, Barbara M. Pearse purified clathrin-coated vesicles from pig brain (Pearse 1975). A year later, she isolated a major protein species of 180 kDa, which she named clathrin “to indicate the lattice-like structures which it forms” (Pearse 1976). It was a breakthrough that inaugurated the molecular dissection of clathrin-mediated endocytosis.Over the intervening years, the continued application of microscopy (which now spans from electron cryotomography to live cell, high-resolution fluorescence microscopy), genetics (in particular, in yeast, Caenorhabditis elegans and Drosophila melanogaster), biochemistry (including cell-free reconstitution of endocytic membrane trafficking events), as well as molecular and structural biology have revealed a great deal about the cellular machineries and mechanisms that govern trafficking along the endocytic pathway. A partial, and because of space limitations, necessarily incomplete list of milestones (

Pfaffosides,nortriterpenoid saponins,from Pfaffia paniculata

Three new nortriterpene saponins having inhibitory effects on the growth of cultured tumor cells, named pfaffosides D, E and F, have been isolated from Pfaffia paniculata. Their structures have been established as 3β-O-[β-d-xylopyranosyl-(1 → 2)-β-d-(6-O-n-butyl) glucuronopyranosyl]-pfaffic acid-(28 → 1)-β-d-glucopyranosyl ester, 3β-O-[β-d-xylopyranosyl-(1 → 2)-β-d(6-O-methyl)glucuronopyranosyl]-pfaffic acid-(28 → 1)-β-d glucopyranosyl ester and 3β-O[β-d-glucuronopyranosyl]-pfaffic acid respectively, based on their chemical and spectroscopic properties     

Structure of sensilla, olfactory perception, and behaviour of the bedbug, Cimex lectularius, in response to its alarm pheromone

When deprived of the terminal antennal segments, male and female bedbugs failed to respond to their alarm pheromone and to their assembling scent. Trans-oct-2-en-1-al or trans-hex-2-en-1-al, being the major constituents of the former, induce in adults and larvae of Cimex lectularius a typical alarm behaviour resulting in dispersal of assembled bedbugs; the rapidity of escape depends on the aldehyde concentration in the air. The behavioural threshold for adults is about 9×10¹⁴ molecules of trans-oct-2-en-1-al or 6×10¹⁵ molecules of trans-hex-2-en-1-al per ml air.The distal part of the terminal antennal segment of C. lectularius reveals the following sensilla: bristles (type A1), immersed cones (type B1), plates (type B2), grooved pegs (type C), smooth pegs (type D), hairs with even (type E1), and uneven wall thickness (type E2). The number and distribution of these sensilla is relatively constant and similar in both sexes, but differs slightly in neonate larvae. The pegs and hairs of types C, D, E1 and E2 were shown to have porous walls, a prerequisite for olfactory function.Receptor potentials were recorded from olfactory sensilla of types E1 and E2 after stimulation with trans-hex-2-en-1-al and trans-oct-2-en-1-al. The minimal concentration of trans-hex-2-en-1-al evoking a receptor potential is about 2×10¹⁰ molecules per ml air. The above olfactory sensilla were found to respond also to hexan-1-al, but almost no responses to pentan-1-al, butan-1-al, trans-hex-2-ene, and trans-oct-2-ene were observed. A minimum chain length of six carbons atoms and a terminal carbonyl group are molecular prerequisites for optimal odorant activity, while the presence of a Δ²-double bond is not essential for stimulation of the alarm pheromone receptors of the bedbug.     

Sheep gene mapping: additional DNA markers included (CASB, CASK, LALBA, IGF-1 and AMH).

DNA extracted from 25 hamster-sheep hybrid cell lines was subjected, after Southern blotting, to hybridization with CASB, CASK, LALBA, IGF-1 and AMH cDNA probes. CASB and CASK segregated together and IGF-1 and LALBA were found syntenic with the LDHB-PEPB-TPI-GAPD-SHMT-KRTB group. No other synteny was observed with any of the previously described groups using the same hybrid cell panel. Gene nomenclature: ACO 1: aconitase 1 (soluble); ADA: adenosine deaminase; AMH: antiMüllerian hormone; ARA 1: murine sarcoma 3611 viral (v-raf) oncogene homologue 1; CASB: beta-casein; CASK: kappa-casein; ENO 1: enolase 1 (alpha); G6PD: glucose-6-phosphate dehydrogenase; GALA (or GLA): glactosidase (alpha); GAPD: glyceraldehyde -3- phosphate dehydrogenase; GPI: glucose phosphate isomerase; GSR: glutathione reductase; HBG: haemoglobin gamma; HPRT: hypoxanthine phosphoribosyl transferase; IDH 1: isocitrate dehydrogenase 1 (soluble); IGF-1: insulin growth factor 1; ITPA: inosine triphosphatase; KRTA: keratin (acid); KRTB: keratin (basic); LALBA: alpha-lactalbumin; LDHA: lactate dehydrogenase A; LDHB: lactate dehydrogenase B; MDH 2: malate dehydrogenase NAD (soluble); ME 1: malic enzyme (soluble); MPI: mannose phosphate isomerase; NP: nucleoside phosphorylase; OLA: ovine leucocyte antigen; OTC: ornitine carbamoyltransferase; PAIS: phosphoribosyl amino imidazole synthetase; PEPA, PEPB, PEPC: peptidase A, B, C; PGD: phospho gluconate dehydrogenase; PGK: phosphoglycerate kinase; PGM 3: phospho glucomutase 3; PKM 2: pyruvate kinase (muscle); PLP: proteolipid protein; PRGS: phosphoribosyl glycinamide synthetase; RCP: red cone pigment; SHMT: serine hydroxymethyl transferase; SOD 1: superoxide dismutase 1 (soluble); SYN 1: synapsin 1; TPI l: triose phosphate isomerase 1.     

光强和氮源及其浓度对缺刻缘绿藻生长、油脂和花生四烯酸积累的影响

对缺刻缘绿藻（Parietochloris incisa（Reisigl） S.Watanabe）在不同光强和氮源及其浓度条件下的生长状况及油脂和花生四烯酸（AA）的积累规律进行了研究。结果显示,缺刻缘绿藻在3种氮源条件下均能较好地生长。在高氮浓度条件下,增大光强能显著提高缺刻缘绿藻的生物量并促进油脂和AA的积累。缺刻缘绿藻在300 μmol·m^-2·s^-1光强、8.8 mmol·L^-1NaNO₃条件下生物量达到最大（4.17 g·L^-1）。油脂含量在100 μmol·m^-2·s^-1光强、1.0 mmol·L^-1氮浓度下达到最高,分别为41.17%（NaNO₃）、42.04%（NH₄HCO₃）和39.96%（CO（NH₂）₂）。AA绝对含量在300 μmol·m^-2·s^-1光强、2.9 mmol·L^-1 NaNO₃条件下达到最高,占细胞干重的16.44%。油脂和AA产率,在300 μmol·m^-2·s^-1光强、以NaNO₃为氮源的条件下达到最大,分别为134.6 mg·L^-1·d^-1（1.0 mmol·L^-1）和35.85 mg·L^-1·d^-1（2.9 mmol·L^-1）。综合考虑成本等因素,选择NH₄HCO₃（5.9 mmol·L^-1）和CO（NH₂）₂（2.9 mmol·L^-1）为氮源、在300 μmol·m^-2·s^-1高光强下培养缺刻缘绿藻进行AA的生产为最优方案。     

Seasonal metabolism of a small, arboreal monitor lizard, Varanus scalaris, in tropical Australia

The field metabolic rates (FMR) and water fluxes of Varanus scalaris were measured during the wet and dry seasons by the doubly-labelled water technique. Seasonal measurements of standard (night-time) metabolism (SMR) and resting (daytime) metabolism (RMR) were made in the laboratory at 18, 24, 30 and 36°C, and maximal oxygen consumption was measured at 36°C on a motorized treadmill. This population was active throughout the year. In the wet season, the mean FMR was 7.8 kJ day⁻¹ (128.0 kJkg⁻¹ day⁻¹; mean mass = 66.4 g, n = 13), and during the dry season the mean was 5.0 kJ day⁻¹ (67.6 kJ kg⁻¹ day⁻¹; mean mass = 77.4 g, n = 17). The mean water flux rates for these animals were 3.6 and 1.2 ml day⁻¹, respectively (60.4 and 16.6 ml kg⁻¹ day⁻¹). The seasonal means of FMR and water flux were significantly different by ANCOVA ( P < 0.0001). Measurements of SMR and RMR were significantly higher in the wet season (ANCOVA: P < 0.0001), but we found no difference in the maximal oxygen consumption between seasons (ANCOVA: P = 0.6). The maximal oxygen consumption of the lizards on the treadmill (2.9 ml min⁻¹= 1.8 ml g⁻¹ h⁻¹), mean mass = 97.4 g, n = 16) was 20 times that of the SMR at the same temperature during the dry season, and 11 times that of the SMR during the wet season. The seasonal differences in FMR were attributable to: changes in SMR (12.2%) and RMR (16.4%); differences in night-time body temperatures (11.3) and daytime body temperatures (16.4%); and activity (broadly defined to include locomotion, digestion, and reproductive costs (43.7%).     

Characterization and potential industrial applications of five novel,thermotolerant, fermentative,yeast strains

All five novel, thermotolerant, alcohol-producing Kluyveromyces marxianus yeast strains, capable of growth at 52°C, grew aerobically on lactose (growth rates of 0.43 to 0.5 h^-1), whey permeate (0.38 to 0.43 h^-1), cellobiose (0.18 to 0.3 h^-1) and xylose (0.18 to 0.26 h^-1). They also grew anaerobically at 40 (0.18 to 0.19 h^-1) and 45°C (0.09 to 0.14 h^-1). All fermented glucose to ethanol and tolerated ethanol at 95 g l^-1 at 45°C. Their potential for industrial alcohol production, possibly in combination with the dairy and wood-related industries, is discussed.I.M. Banat was and R. Marchant is with the School of Applied Biology and Chemical Sciences, University of Ulster. Coleraine BT52 1SA, UK. I.M. Banat is now with the Department of Biology, UAE University, P.O. Box 17551. Al Ain. Abu-Dhabi, United Arab Emirates