Equilibrium density-gradient procedure for selection of synchronous cells from asynchronous cultures.

A reproducible isopycnic technique, in which linear density gradients of Ficoll are used, was developed for selecting synchronous daughter cells from asynchronous cultures of Chlorella for biochemical studies of the cell cycle.

The role of glycolipids in mediating cell adhesion: a flow chamber study.

Selectins constitute a family of proteins that mediate leukocyte tethering and rolling along the vascular endothelium by recognizing various carbohydrate ligands in response to inflammation. To test the hypothesis that multivalent binding of selectins to their ligands is the molecular basis for achieving sufficient binding forces, we have performed this flow chamber study. Selectin-containing Chinese hamster ovarial cells (CHO-E) bind and roll along a support-fixed phospholipid membrane containing a defined concentration of a synthetic Sialyl Lewisx (sLex) glycolipid ligand. Ligands are either homogeneously distributed, or arranged in defined lateral clusters, as illustrated here for the first time. The lateral glycolipid clusters which appear as recognition motifs are essential for mediating cell rolling. Furthermore, the transition from firm cell adhesion to cell rolling depends on the site density of ligands. Rolling velocity shows little dependence on shear forces within a broad range. As we found out that cells do not roll along the model membranes with homogeneous ligand distribution, our results therefore support the hypothesis of multivalent binding events. Since these investigations suggest that lipid-anchored sLex, functionally embedded in a lipid matrix, can mediate cell rolling, this study demonstrates the relationship between dynamic glycolipid binding to selectins with the hypothesis of multivalency of binding for the first time.

Coordinate and non-coordinate accululation of aspartate transcarbamylase and dihydroorotase in synchronous Chlorella cells growing on different nitrogen sources.

Regulation of the levels of aspartate transcarbamylase (carbamoylphosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) and dihydroorotase (L-5,6-dihydro-orotate amidohydrolase, EC 3.5.2.3) was studied in synchronous cultures of the eucaryotic microorganism Chlorella. Analytical polyacrylamide gel electrophoresis and sucrose density-gradient centrifugation studies revealed that these cells contain a single aspartate transcarbamylase and a dihydroorotase with apparent molecular weights of 160 000 and 80 000, respectively. In synchronous cells cultured in nitrate medium, these two enzymes accumulated in single step-patterns over different periods of the cell cycle. In contrast, these enzymes accumulated in a coordinate manner throughout the cell cycle in ammonium medium. Experiments with inhibitors of protein and RNA synthesis indicated that dihydroorotase is stable in vivo and suggested that cell cycle changes in the turnover rate of aspartate transcarbamylase might determine whether or not these enzymes accumulate in a coordinate manner. Although uracil and uridine could be absorbed and metabolized by the cells, synthesis of these two enzymes could not be repressed by culturing synchronous cells in medium, containing high concentrations (29-40 mM) of uracil or uridine, for an entire cell cycle.

The beta-bungarotoxin-binding protein from chick brain: binding sites for different neuronal K+ channel ligands co-fractionate upon partial purification.

beta-Bungarotoxin (beta-Butx) is a presynaptically active neurotoxin which blocks neuronal A-type K+ channels. Here, the efficient solubilisation and about 300-fold purification of the beta-Butx-binding protein from chick brain were achieved by detergent extraction at high ionic strength followed by chromatography on DEAE Affigel Blue, beta-Butx Affigel 102 and wheat germ agglutinin Sepharose. Binding of 125I-labelled beta-Butx to the purified protein was inhibited by two other K+ channel ligands, dendrotoxin I and mast cell-degranulating peptide. It is concluded that the beta-Butx-binding protein is a member of a family of voltage-gated K+ channels which exhibit varying affinities for different polypeptide ligands.

Sphingomyelin is synthesized in the cis Golgi.

We have employed in vitro a truncated ceramide analogue with 8 carbon atoms in the sphingosine and the fatty acyl residue, each, to investigate the activity of various membrane fractions to synthesize truncated sphingomyelin. This shortened ceramide readily diffuses through membranes and therefore can easily find access to the lumina of intact organelles. Sphingomyelin synthase activity resides in the Golgi apparatus, and after sucrose density gradient centrifugation of Golgi-enriched fractions sphingomyelin synthesis follows a cis Golgi marker enzyme.

Differential effects of synthetic sphingosine derivatives on melanoma cell motility, growth, adhesion and invasion in vitro.

Cancer cell surface glycosphingolipids are considered to play a critical role in tumor growth and metastasis. However, the implications of glycoconjugates in the control of cell motility, which is considered to be involved in tumor invasion, are not fully understood. In this study, the effects of a series of synthetic sphingosine derivatives, obtained by the chemical transformation of azidosphingosines, on directional migration of K1735-M2 melanoma cells grown on type I collagen-coated surfaces were investigated. Following the application of 60 microM (2R, 3S, 4E)-2, 3-epimino-4-octadecen-3-ol (S4) the migration rate was 94 +/- 10 microns/day, compared with 377 +/- 22 microns/day in the control experiment. Six other analogues were not as potent. S4 also considerably down-modulated melanoma single cell motility. Inhibition of motile activity was associated with changes in the actin filament organization as well as with changes in the number and distribution of vinculin plaques. Moreover, the compound reduced the attachment abilities of melanoma cells to basement membrane Matrigel. Tumor cell invasion, however, was less affected and proliferation remained unimpaired after treatment with S4. These data suggest at least one intracellular mode of action of this particular synthetic sphingosine derivative by modulation of cytoskeletal organization. Melanoma cell motility and growth may be controlled independently via glycosphingolipids.

Factors involved in immunization program for swine influenza.

The decision to undertake a nationwide program of vaccination against swine influenza requires assessment of the status of immunity of those in various age groups in our population against this agent. Pools of serum were collected from persons born in the years from 1889 to 1943; they were tested for hemaggultinin inhibiting (HI) antibody against the HSW 1N1 influenza virus strains isolated in 1931 and 1976. The titers secured serve as an indication of the average level of immunity of those of different ages. Persons less than 43 years of age are found to be without antibody protection. The need for vaccination of people in different age groups based on mortality statistics of previous epidemics is evaluated. It is realized that no epidemic may occur and that a reduced virulence of the viral agent and use of antibiotics may reduce the death rate if the infection recurs. The extraordinary high mortality in 1918 in people between 15 and 44 years of age deserves recognition together with the fact that those in the same age group are now without protection. The fact that women of childbearing age fall into this group deserves special consideration in view of increased mortality in puerperal women observed in the pandemics of 1918 and 1957. The degree of protection afforded the newborn by transplacental transmission of maternal antibodies is discussed. The need of increasing the level of immunity in those who have varying titers of HI antibodies is considered in relation to the prevalence of cardiopulmonary complications and other chronic diseases in older subjects.

Neonatal herpes simplex infection possibly acquired via maternal breast milk.

A newborn infant with disseminated herpes simplex virus type 1 (HSV-1) infection was determined serologically to have acquired the infection postnatally; his mother was found to have HSV-1 in her breast milk but had no history of genital lesions and negative viral cultures of cervix, vagina, and throat. We suggest that HSV-infected maternal breast milk may be a source of this infection for susceptible infants.

A new phenoxyacetate-based linker system for the solid-phase synthesis of oligosaccharides.

[structure: see text]. A novel linker system has been designed, and its first application to solid-phase oligosaccharide synthesis is described. The use of the highly reactive o-nitro-phenoxyacetate linker allows a fast and quantitative cleavage using mild basic conditions. This method combined with the trichloroacetimidate glycosylation exhibits highly promising results as demonstrated for the synthesis of tetrasaccharide 1 (n = 3) containing glucose beta(1 --> 4) and beta(1 --> 6) linkages.

O-Glycosyl trichloroacetimidates bearing Fmoc as temporary hydroxy protecting group: a new access to solid-phase oligosaccharide synthesis.

Different O-glycosyl trichloroacetimidates bearing base sensitive Fmoc protected hydroxy groups were efficiently prepared with CCl(3)CN using a catalytic amount of sodium hydride. The resulting glycosyl donors were engaged in glycosylation reactions both in solution and on solid support with a new ester-type linker with good results. In both approaches, Fmoc groups were afterward quantitatively cleaved using mild basic conditions.

Strategy for efficient detection of respiratory viruses in pediatric clinical specimens.

Direct immunofluorescence (IF) with a polyclonal respiratory syncytial virus (RSV)-specific antibody preparation was used for antigen detection during the 1982-1983 RSV season (155 specimens) and gave an overall sensitivity of 94% with 87% specificity compared with viral culture. Indirect IF was used in the 1983-1984 season (265 specimens) and exhibited sensitivity of 96% with specificity of 79%. During these two seasons, 42 of 224 (18.8%) specimens that were IF-negative for RSV grew viruses other than RSV. In the winter of 1984-1985, we screened 297 specimens for RSV by IF and 80 (27%) were positive. Forty-four (20%) of the IF-negative specimens were culture-positive for RSV(2) or other viruses(44). We conclude that, in the interest of cost reduction and expeditious detection of respiratory viruses, once a properly equipped laboratory has become thoroughly familiar with IF techniques, pediatric respiratory specimens can be screened for RSV by IF and only the IF-negative specimens need be inoculated into cell cultures for isolation of virus during the winter respiratory season.

Recording urethral pressure profile: comparison of methods and clinical implications.

Urodynamic evaluation of urethral sphincter efficiency can be carried out by constant carbon dioxide perfusion, constant water perfusion, or the use of membrane catheters. A comparison of these three techniques was made in in vitro and in vivo experiments. Results, and advantages and disadvantages of each method are discussed. From all factors considered in this study--responsiveness to pressure changes sensitivity to pressures at various levels along the urethra, and reproducibility and accuracy of the profile--the membrane catheter system was by far superior to either of the flow systems.

Surgical anatomy of the vertebral arteries.

STUDY DESIGN: This study compared direct measurements of the distances between the vertebral arteries in the cervical spines of human cadaver specimens with data obtained from axial computed tomography images of these specimens. OBJECTIVE: To determine whether the information obtained from a computed tomography scan can be used reliably to predict the true anatomic location of the vertebral arteries and, in so doing, provide accurate guidelines for the lateral extent of anterior cervical decompressive procedures. SUMMARY OF BACKGROUND DATA: Iatrogenic vertebral artery injury during anterior cervical surgery is uncommon, potentially catastrophic, and avoidable. METHODS: The means and standard deviation of measurements of the location of the cervical segment of the vertebral arteries obtained with high-precision, digital calipers by direct gross anatomic dissection of 16 adult (eight male, eight female) cadaver specimens were recorded. These measurements were compared with computed tomography scan data obtained on the same specimens. RESULTS: The mean distances between the vertebral arteries progressively increased from C3 to C6. Computed tomography scan measurements of the distance between the cervical foramina transversaria were consistently smaller than direct measurements of the gross specimens. At C6, the computed tomography scan data were significantly less than the gross anatomic data. CONCLUSIONS: According to these data, computed tomography scan measurements may be used safely and accurately to plan the lateral extent of anterior cervical decompressive surgical procedures. Although the data obtained from the gross anatomic dissections may serve as guidelines to assist the surgeon, the authors recommend a careful review of the preoperative computed tomography scan on an individual case-by-case basis as the safest method to plan for anterior cervical surgery.

A convenient synthesis of N-acetyllactosamine derivatives from lactal.

In a thermal inverse-type hetero-Diels-Alder reaction of O-silyl-protected lactal 1 and bis(2,2,2-trichloroethyl) azodicarboxylate (2), the dihydrooxadiazine derivative 3 was obtained in a very high yield; transesterification with benzyl alcohol furnished the corresponding derivative 4. Treatment of 3 with methanol in the presence of BF3.OEt2 afforded the methyl lactoside derivative 5 which, after transesterification with benzyl alcohol, then hydrogenolytic debenzylation and concomitant NN-cleavage with Raney nickel, and N-acetylation, furnished methyl O-(2,4,6-tri-O-tert-butyldimethylsilyl-beta-D-galactopyranosyl)-(1-->4)- 2-acetamido-3,6-di-O-tert-butyldimethylsilyl-2-deoxy-beta-D-glucopyra nos ide (7) in high yield. Desilylation of 4, then O-acetylation, methyl glycoside formation with methanol-BF3.OEt2, hydrogenolytic debenzylation, and NN-cleavage with Raney nickel, and N-acylation afforded methyl O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->4)-2-acetamido-3 ,6-di-O-acetyl-2-deoxy-beta-D-glucopyranoside (10).

Application of the trichloroacetimidate method to the synthesis of glycopeptides of the mucin type containing a beta-D-Galp-(1----3)-D-GalpNAc unit.

Mucin-type O-glycopeptides containing the beta-D-Galp-(1----3)-D-GalpNAc disaccharide core unit, which is also the T-antigenic determinant, were synthesized from D-galactose, 2-azido-2-deoxy-D-galactose, 2-azido-2-deoxylactose, and L-serine precursors by applying the trichloroacetimidate method. Thus, beta-D-Galp-(1----3)-alpha-D-GalpNAc-(1----3)-Ser (1) and beta-D-Galp-(1----4)-beta-D-GlcpNAac-(1----6)-[beta-D-Galp-(1----3 )]-alpha-D-GalpNAc-(1----3)-Ser (2) were obtained. A protected precursor of 2 having free OH groups at C-4 and C-6 of the inner sugar unit is a valuable intermediate for the synthesis of further O-glycopeptides of this core-unit type.

Synthesis of Kdo-alpha-glycosides of lipid A derivatives.

The synthesis of the lipopolysaccharide fragment O-(4,5,7,8-tetra-O-acetyl-3-deoxy-N-methyl-alpha-D-manno-2- octulopyranosylonamide)-(2-->6)-O-(2-deoxy-2-[(3R)-3- dodecanoyloxytetradecanamido]-4-O- phosphono-3-O-tetradecanoyl-beta-D-glucopyranosyl)-(1-->6)-1-O-acetyl-2- deoxy-2 - [(3R)-3-dodecanoyloxytetradecanamido]-3-O-tetradecanoyl-alpha-D- glucopyranose (35 alpha) is performed via anomeric O-alkylation. With this objective, the 2-azido-3-O-benzyl-2-deoxy-6-O-trifluoromethanesulfonyl-beta-D-glu copyranosides 5, 7, and 19 alpha, beta were synthesized from D-glucal and employed as alkylating agents. Reaction of 5 with the O-cyclohexylidene-protected Kdo-derivative 10 afforded the desired alpha-linked disaccharide, tert-butyldimethylsilyl 4-O-allyl-2-azido-3-O-benzyl-2-deoxy-6- O-(4,5:7,8-di-O-cyclohexylidene-3-deoxy-N-methyl-alpha-D-manno-2- octulopyranosylonamide)-beta-D-glucopyranoside (11); even better yields of the structurally related disaccharide 12 were obtained with the 4-O-unprotected 7 as alkylating agent. 1-O-Desilylation of 12 furnished the lactol 20, which could be alkylated at the anomeric position with 1-O-allyl protected alkylating agents 19 alpha and 19 beta, both of which furnished exclusively the desired beta-(1-->6)-linked trisaccharides allyl O-(4,5:7,8-di-O-cyclohexylidene-3- deoxy-N-methyl-alpha-D-manno-2-octulopyranosylonamide)-(2-->6)-O-( 2-azido-3- O-benzyl-2-deoxy-beta-D-glucopyranosyl)-(1-->6)-2-azido-3, 4-di-O-benzyl-2-deoxy-alpha- (21 alpha) and -beta-D-glucopyranoside (21 beta), respectively. Phosphorylation with diphenyl phosphorochloridate, replacement of the O-cyclohexylidene protective group by O-triethylsilyl (TES) protective groups, removal of the 1-O-allyl group, azido group reduction, subsequent N-acylation, and then O-acetylation provided the key 1-O-acetyl protected intermediate 30 alpha. Removal of the O-TES groups, subsequent O-acetylation, and hydrogenolytic O-debenzylation furnished O-[4,5:7,8-tetra-O-acetyl-3-deoxy-N-methyl-alpha-D-manno-2- octulopyranosylonamide]-(2-->6)-O-(2-deoxy-4-O-diphenoxyphospho ryl-2-[(3R)- 3-dodecanoyloxytetradecanamido]-beta-D-glucopyranosyl)-(1-->6)-1-O -acetyl-2- deoxy-2[(3R)-dodecanoyloxytetradecanamido]-alpha-D-glucopyranose (33 alpha), which underwent the required selective O-tetradecanoylation at the 3-O- and 3'-O-position, thus furnishing, after hydrogenolytic O-dephenylation of the diphenoxyphosphoryl group, the target molecule 35 alpha.

Solid-phase synthesis of a glycosylated hexapeptide of human sialophorin, using the trichloroacetimidate method.

A hexapeptide containing a beta-D-Gal p-(1-->3)-alpha-D-Gal pNAc-(1-->O)-L-threonine unit was synthesized using glycosylated pentafluorophenyl esters in an Fmoc-based strategy. In all of the glycosylation reactions, trichloroacetimidates were successfully employed. The disaccharide moiety was prepared from tetra-O-acetyl-alpha-D-galactopyranosyl trichloroacetimidate and tert-butyldimethylsilyl 2-azido-6-O-benzoyl-2-deoxy-beta-E-galactopyranoside with boron trifluoride etherate as a catalyst. The glycosylated active esters were obtained in the reaction of alpha and beta 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1-->3)-4-O-acetyl-2-azid o-6- O-benzoyl-2-deoxy-D-galactopyranosyl trichloroacetimidates with Fmoc-protected pentafluorophenyl esters of L-serine and L-threonine in the presence of trimethylsilyl trifluoromethanesulfonate as Lewis acid. The glycosylated pentafluorophenyl ester of L-threonine was transformed into glycopeptides via a solid-phase synthesis. Azide reduction and N-acetylation were performed on the solid phase with a thioacetic acid-pyridine mixture. The glycopeptide was then cleaved from the resin with strong acid, also removing the acid-labile protecting groups of the peptide chain. Finally, the acyl groups used for sugar protection were cleaved with sodium methoxide, affording the completely deprotected N-acetyl-L-leucyl-L-glutamyl-O-[beta-D-galactopyranosyl-(1-->3)-2- acetamido-2-deoxy-alpha-D-galactopyranosyl]-L-threonyl-L-seryl-L-threony l- glycinamide (1) in high purity.

Total synthesis of alpha-galactosyl cerebroside.

A highly convergent synthetic approach was developed to obtain alpha-galactosyl cerebroside O-(alpha-D-galactopyranosyl)-2-hexacosylamino-D-ribo-1,3,4-octa decantriol, which has previously been demonstrated to have immunostimulatory activity. Known 4,6-O-benzylidene galactose was the starting material for both the required alpha-galactosyl and the phytosphingosine building blocks O-(2,3-di-O-benzyl-4,6-O-benzylidene-D-galactopyranosyl) trichloroacetimidate (4) and 2-O-methanesulfonyl-D-arabino-1,2,3,4-octadecantetrol (5). The key step of the synthetic strategy is the highly regio- and stereoselective O-galactosylation of 1,3,4-O-unprotected phytosphingosine acceptor 5 using known 4 as donor. The total synthesis required only 11 synthetic steps starting from galactose.

Muramic acid derivatives as glycosyl donors for the synthesis of muramyl-containing glycosphingolipids and fatty acids.

2-Azido-2-deoxy-4,6-O-isopropylidene-3-O-[(1R)-(methoxycarbonyl)ethyl]- alpha-D-glucopyranosyl trichloroacetimidate (3 alpha) has been used as the glycosyl donor in the synthesis of glycosphingolipids 14 and 27. Reaction of 3 alpha with (2S, 3R, 4E)-2-azido-3-benzoyloxy-4-octadecen-1-ol (6) gave (2S, 3R, 4E)-2-azido-1-(2-azido-2-deoxy-4,6-O-isopropylidene-3-O-[(1R)-1-(m ethoxycarbonyl)ethyl]-beta-D-glucopyranosyloxyl)-3-benzoyloxy-4- octadecene (7), which was converted into (2S, 3R, 4E)-1-(2-deoxy-2-hexadecanoylamino-3-O-[(2R)-propanoyl-(L-alanyl-D -isoglutamine benzyl ester)-2-yl]-beta-D-glucopyranosyloxy)-2-hexadecanoylamino-4-oc tadecen-ol (14). Reaction of 3 alpha with tert-butyldimethylsilyl 2-azido-3,6-di-O-benzyl-2-deoxy-beta-D-glucopyranoside (15) gave tert-butyldimethylsilyl 2-azido-4-O-(2-azido-2-deoxy-4,6-O-isopropylidene-3-O-[(1R)-1-(methox ycarbonyl)ethyl]-beta-D-glucopyranosyl)-3,6-di-O-benzyl-2-deoxy-be ta- D-gluc opyranoside (16 beta), which was converted into 1,3,6-tri-O-acetyl-2-deoxy-4-O-(4,6-di-O-acetyl-2-deoxy-2-hexadecanoy lam ino-3-O-[2R)-propanoyl-(L-alanyl-D-isoglutamine methyl ester)-2-yl]-beta-D-glucopyranosyl)-2-hexadecanoylamino-D-glucopyranose (27).