Robotic transanal minimally invasive surgery (TAMIS) for local excision of rectal lesions with the da Vinci Xi (dVXi): technical considerations and video vignette.

Robotic transanal minimally invasive surgery (TAMIS) (RT) represents a compelling new alternative capable of overcoming the limitations of conventional TAMIS for the local excision of rectal lesions. We describe our RT technique using the dVXi™ (Intuitive Surgical, Sunnyvale, CA, USA) which we have used to efficiently and completely excise eight cases of rectal lesions which were not endoscopically resectable. We also include a video vignette of the procedure. With the patient in the prone jackknife position, we insert a GelPOINT™ Path Transanal Access Platform (Applied Medical, Rancho Santa Margarita, CA, USA) in combination with the dVXi and AirSeal™ insufflation system (Conmed, Niagara. Falls, ON, Canada). Our technique aims to be ergonomically efficient to minimise docking difficulties and to reduce instrument clash in the limited space, whilst maximising the capabilities of the dVXi for RT. At 3-month endoscopic follow-up, no evidence of recurrence was detected in any of the eight patients. RT is safe, feasible and has advantages over conventional laparoscopic TAMIS (LT). Our described technique addresses some of the long-standing challenges of LT and the novel RT. The immediate challenge to its widespread use remains the cost, expertise and availability.

Pharmacokinetics of enrofloxacin and ceftiofur in plasma, interstitial fluid, and gastrointestinal tract of calves after subcutaneous injection, and bactericidal impacts on representative enteric bacteria.

This study's objectives were to determine intestinal antimicrobial concentrations in calves administered enrofloxacin or ceftiofur sodium subcutaneously, and their impact on representative enteric bacteria. Ultrafiltration devices were implanted in the ileum and colon of 12 steers, which received either enrofloxacin or ceftiofur sodium. Samples were collected over 48 h after drug administration for pharmacokinetic/pharmacodynamic analysis. Enterococcus faecalis or Salmonella enterica (5 × 10(5) CFU/mL of each) were exposed in vitro to peak and tail (48 h postadministration) concentrations of both drugs at each location for 24 h to determine inhibition of growth and change in MIC. Enrofloxacin had tissue penetration factors of 1.6 and 2.5 in the ileum and colon, while ciprofloxacin, an active metabolite of enrofloxacin, was less able to cross into the intestine (tissue penetration factors of 0.7 and 1.7). Ceftiofur was rapidly eliminated leading to tissue penetration factors of 0.39 and 0.25. All concentrations of enrofloxacin were bactericidal for S. enterica and significantly reduced E. faecalis. Peak ceftiofur concentration was bactericidal for S. enterica, and tail concentrations significantly reduced growth. E. faecalis experienced growth at all ceftiofur concentrations. The MICs for both organisms exposed to peak and tail concentrations of antimicrobials were unchanged at the end of the study. Enrofloxacin and ceftiofur achieved intestinal concentrations capable of reducing intestinal bacteria, yet the short exposure of ceftiofur in the intestine may select for resistant organisms.

Correction of a defect in mammalian GPI anchor biosynthesis by a transfected yeast gene.

Glycosylphosphatidylinositol (GPI) serves as a membrane anchor for a large number of eukaryotic proteins. A genetic approach was used to investigate the biosynthesis of GPI anchor precursors in mammalian cells. T cell hybridoma mutants that cannot synthesize dolichol-phosphate-mannose (Dol-P-Man) also do not express on their surface GPI-anchored proteins such as Thy-1 and Ly-6A. These mutants cannot form mannose-containing GPI precursors. Transfection with the yeast Dol-P-Man synthase gene rescues the synthesis of both Dol-P-Man and mannose-containing GPI precursors, as well as the surface expression of Thy-1 and Ly-6A, suggesting that Dol-P-Man is the donor of at least one mannose residue in the GPI core.

Neurovisceral and skeletal GM1-gangliosidosis in dogs with beta-galactosidase deficiency.

Beta-galactosidase-deficient siblings in two litters of English springer spaniel puppies showed a progressive neurological impairment, dwarfism, orbital hypertelorism, and dysostosis multiplex. An excess of GM1-ganglioside was found in the brain. Three abnormal oligosaccharides were present in samples of urine, brain, liver, and cartilage. Light microscopy of selected tissue specimens revealed cytoplasmic vacuoles in neurons, circulating blood cells, macrophages, and chondrocytes. Ultrastructural studies demonstrated that these membrane-bound vacuoles were of two types--one containing lamellated membranes and the other, finely granular material. These clinical and pathological findings are similar to those observed in human patients affected by the infantile form of GM1-gangliosidosis.

Differential expression of glycosylphosphatidylinositol-anchored proteins in a murine T cell hybridoma mutant producing limiting amounts of the glycolipid core. Implications for paroxysmal nocturnal hemoglobinuria.

A T cell hybridoma mutant, which expressed a markedly reduced level of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell surface, was characterized. The surface expression level of Thy-1 was approximately 17% of the wild-type level, whereas the surface expression of Ly-6A was approximately 2.4% of the wild-type level. We show here that these cells synthesized limiting amounts of the GPI core and that the underlying defect in these cells was an inability to synthesize dolichyl phosphate mannose (Dol-P-Man) at the normal level. The defect in Ly-6A expression could be partially corrected by tunicamycin, which blocked the biosynthesis of N-linked oligosaccharide precursors and shunted Dol-P-Man to the GPI pathway. Full restoration of Thy-1 and Ly-6A expression, however, required the stable transfection of a yeast Dol-P-Man synthase gene into the mutants. These results revealed that when the GPI core is limiting, there is a differential transfer of the available GPI core to proteins that contain GPI-anchor attachment sequences. Our findings also have implications for the elucidation of the defects in paroxysmal nocturnal hemoglobinuria.

Solubilization of an alpha-(1 leads to 3)-D-mannosyltransferase from pancreas which utilizes synthetic dolichyl pyrophosphate trisaccharide beta-Man-(1 leads to 4)-beta-GlcNAc-(1 leads to 4)GlcNAc as substrate.

Calf pancreas microsomes were treated with 0.5-1% Triton X-100 and the resulting soluble enzyme preparation was incubated with GDP-D-[14C]mannose. The addition of synthetic Dol-PP derivative of the trisaccharide beta-Man-(1 leads to 4)-beta-GlcNAc-(1 leads to 4)GlcNAc stimulated the synthesis of labeled lipid-bound tetrasaccharide 50-100-fold. The labeled tetrasaccharide thus formed was identified as alpha-Man-(1 leads to 3)-beta-Man-(1 leads to 4)-beta-GlcNAc- (1 leads to 4)GlcNAc by its chromatographic properties and by its sensitivity to alpha-mannosidase and to endo-beta-N-acetylglucosaminidase D. The solubilized alpha-(1 leads to 3)mannosyltransferase did not require divalent cation and was active in the presence of 10 mM EDTA.

Characterization of oligosaccharides from the urine of loco-intoxicated sheep.

Two major oligosaccharides were isolated by preparative HPLC from the urine of a locoweed-fed sheep. Analysis by gas-liquid chromatography and mass-spectrometry indicated compositions of (Man)4(GlcNAc)2 and (Man)5(GlcNAc)2, respectively. Structures were determined by digestion with alpha-D-mannosidase and endo-beta-N-acetylglucosaminidases D and H, and comparison of the products by HPLC with synthetic standards, and oligosaccharides isolated from human mannosidosis urine. Incubation with an exo-beta-N-acetylglucosaminidase was without effect.

Induced mannosidosis-excretion of oligosaccharides by locoweed-intoxicated sheep.

Daily urine samples were collected from a locoweed-fed sheep, and the oligosaccharide content examined by thin-layer and liquid chromatography. An unusual pattern of urine oligosaccharides was observed, which appears to be characteristic of loco intoxication. Changes in the pattern could be correlated with the onset of visible disease, which occurred approximately 5 weeks after the typical urine sugars were first detected. HPLC showed that these sugars consisted of two homologous series of oligosaccharides containing one and two residues of 2-acetamido-2-deoxy-D-glucose, respectively.

Oligosaccharides from placenta: early diagnosis of feline mannosidosis.

High-pressure liquid chromatography analysis of oligosaccharides from placentas allowed the diagnosis of alpha-mannosidosis in three litters of kittens. The chromatography also afforded a detailed comparison of the oligosaccharide pattern and levels in placenta, liver, brain, urine and ocular fluid of the affected animals. In all cases, two series of compounds were observed, with one or two residues of N-acetylglucosamine at the reducing terminus, respectively, and between two and nine mannose residues. This pattern is unlike that of human mannosidosis, and resembles that of ruminants, except that the major oligosaccharide contains three mannose residues instead of two.

Biochemical, ultrastructural and histochemical studies of cat placentae deficient in activity of lysosomal alpha-mannosidase.

Lysosomal alpha-mannosidase activity, oligosaccharide profiles, light and electron microscopy and lectin histochemistry studies were performed on full-term placentae obtained from five litters of cats. They resulted from breeding related cats who are obligate heterozygotes for lysosomal alpha-mannosidase deficiency. alpha-Mannosidase activity in placentae from affected kittens was less than 10 per cent of control, while in placentae from presumptive heterozygotes the activity was less than 50 per cent of control. High-pressure liquid chromatographic analysis of oligosaccharides revealed massive accumulation of undegraded oligosaccharides in placentae of affected kittens. A small elevation was found in placentae from presumptive heterozygous kittens, and none was detected in placentae of normal kittens. Light and electron microscopic examinations revealed vacuolization of fetal endothelial and mesenchymal cells only in placentae of affected kittens. Succinylated wheat germ agglutinin and concanavalin A stained the fetal fibroblasts only in placentae of affected kittens.

Resolution of structural isomers of sialylated oligosaccharides by capillary electrophoresis.

The resolution of structural isomers in mixtures of oligosaccharides is often challenging. Capillary electrophoresis was employed to separate three sets of structural isomers of sialylated oligosaccharides found in human milk and bovine colostrum. Different running buffers were necessary to achieve optimal baseline resolution. To resolve 3'- and 6'-sialyllactoses, 0.2 M aqueous sodium phosphate containing 40% methanol as an organic modifier was used as a running buffer. To resolve 3'- and 6'-sialyllactosamines, 0.4 M aqueous sodium phosphate without organic modifier was used. Baseline resolution of sialyllacto-N-tetraose-a and -b and sialyllacto-N-neotetraose-c was achieved with a 0.4 M Tris-HCl buffer containing 250 mM sodium dodecyl sulfate and 10% methanol as the organic modifier. Thus, each of these sets of structural isomers of sialylated oligosaccharides required a unique running buffer with respect to buffer type, concentration, pH, presence of organic modifiers, and surfactants. Similar electrophoresis conditions may be useful for resolving and analyzing other structural isomers of acidic oligosaccharides by capillary electrophoresis.

The synthesis of allyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-alpha-D-glucopyranoside and of chitobiose derivatives by the oxazoline procedure.

Controlled, partial benzylation of allyl 2-acetamido-3-O-benzyl-2-deoxy-alpha-D-glucopyranoside gave a mixture of the 3,4-di-, 3,6-di- (15), and 3,4,6-tri-O-benzyl derivatives, the major product being 15. Condensation of 15 with 2-methyl-(3,4,6-tri-O-acetul-1,2-dideoxy-alpha-D-glucopyrano)-[2,1-d]-2-oxazoline gave a disaccharide which, after purification, removal of the allyl group, and hydrogenolysis of the benzyl substituents, gave 2-acetamido-4-O-(2-acetamido-3,4,6-tri-O-acetyl-i-deoxy-beta-D-glucopyranosyl)-2-deoxy-alpha-D-glucopyranose. This compound was further converted into di-N-acetyl-hexa-O-acetylchitobiose by acetylation, or into 2-methyl-[4-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-3,6-di-O-acetyl-1,2-di-deoxy-alpha-D-glucopyrano]-[2,1-d]-2-oxazoline, a starting material for the preparation of di-N-acetyl-alpha-chitobiosyl phsophate.

Application of lectin histochemistry and carbohydrate analysis to the characterization of lysosomal storage diseases.

In lysosomal storage diseases that involve a defect in the catabolism of glycoconjugates, lectin histochemistry adds a new dimension to the characterization of stored carbohydrates as it identifies sugar residues in situ in the affected cells and, thus, determines which cell types are affected by storage. It may be combined with chemical and biochemical analysis by h.p.l.c. The present review summarizes recent results for a variety of storage diseases and presents new data for GM1-gangliosidosis.

The preparation of a partially protected heptasaccharide-asparagine intermediate for glycopeptide synthesis.

The heptasaccharide O-alpha-D-mannopyranosyl-(1----6)-O-[alpha-D-mannopyranosyl-(1----3)]-O- alpha-D-mannopyranosyl-(1----6)-O-[alpha-D-mannopyranosyl-(1----3)]-O-be ta- D-mannopyranosyl-(1----4)-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)- (1----4)-2-acetamido-2-deoxy-D-glucopyranose, isolated from the urine of swainsonine-intoxicated sheep, was peracetylated and was converted into the glycosyl azide by three alternative procedures, the most successful of which was formation of peracetyl oxazoline by treatment with trimethylsilyl trifluoromethanesulfonate, followed by treatment with trimethylsilyl azide. Reduction of the glycosyl azide in the presence of Lindlar catalyst gave the glycosylamine derivative, which was coupled with 1-benzyl N-fluoren-9-ylmethoxycarbonyl-L-aspartate to yield a protected glycosylasparagine. The benzyl ester group was easily removed by hydrogenolysis to form an intermediate suitable for glycopeptide synthesis.

Synthesis of the tetrasaccharide lipid intermediate P1-dolichyl P2-[O-alpha-D-mannopyranosyl-(1----6)-O-beta-D-mannopyranosyl-(1----4) -O-(2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-2- deoxy-alpha-D-glucopyranosyl] diphosphate.

O-alpha-D-Mannopyranosyl-(1----6)-O-beta-D-mannopyranosyl-(1----4)-O-(2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-2-deoxy- D-glucopyranose was isolated from bovine or ovine mannosidosis urine. After peracetylation, treatment with trimethylsilyl trifluoromethanesulfonate gave a high yield of a peracetyl oxazoline, which was phosphorylated with dibenzyl phosphate to give a dibenzyl glycosyl phosphate that was converted into a peracetyl tetrasaccharide phosphate by catalytic hydrogenolysis. A coupling reaction with P1-dolichyl P2-diphenyl diphosphate, prepared in two stages from pig-liver dolichol, yielded a peracetyl diphosphoric diester, which on O-deacetylation gave P1-dolichyl P2-[O-alpha-D-mannopyranosyl-(1----6)-O-beta-D-mannopyranosyl-(1----4)-O -(2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-2-deoxy- alpha-D- glucopyranosyl] diphosphate. This tetrasaccharide lipid intermediate was active as an acceptor of D-mannose residues from GDP-D-mannose in the presence of calf pancreas microsomes.

Chromatographic separation of oligosaccharides from mannosidosis urine.

A mixture of oligosaccharides was isolated from mannosidosis urine by a rapid and convenient procedure employing adsorption on activated charcoal. The mixture was partially fractionated into a homologous series of compounds by a rapid procedure employing a preparative, liquid chromatograph, and a more complete separation was obtained by a second chromatographic step employing a solid phase having more-powerful resolving properties, or by preparative-layer chromatography. The series of oligosaccharides was completely separated by 7-MPa, liquid chromatography (l.c.) on a Micropak NH2-10 column; the analysis could be performed with isocratic or gradient solvent-systems, and did not involve derivatization. With the isocratic system, a strict relationship was observed between retention time and the number of D-mannosyl residues. The use of 1,4-diaminobutane as a column "restorer" was evaluated.

The synthesis of a trisaccharide and a tetrasaccharide lipid intermediate. P1-dolichyl P2-[O-beta-D-mannopyranosyl-(1----4)-O-(2-acetamido-2-deoxy- beta-D-glucopyranosyl)-(1----4)-2-acetamido-2-deoxy-alpha-D- glucopyranosyl] diphosphate and P1-dolichyl P2-[O-alpha-D-mannopyranosyl-(1----3)-O-beta-D- mannopyranosyl-(1----4)-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)- (1----4)-2-acetamido-2-deoxy-alpha-D-glucopyranosyl] diphosphate.

Synthetic benzyl O-(2,3,4,6,-tetra-O-acetyl-beta-D-mannopyranosyl)-(1-4)- (2-acetamido-3,6-di-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-(1-4)- 2-acetamido-3,6-di-O-benzyl-2-deoxy-alpha-D-glucopyranoside was converted, by catalytic hydrogenolysis of the benzyl groups, followed by treatment with acetyl chloride-hydrogen chloride and then "chloride-ion catalysis", into O-(2,3,4,6-tetra-O-acetyl- beta-D-mannopyranosyl)-(1-4)-O-(2-acetamido-3,6-di-O-acetyl-2-deoxy beta- D-glucopyranosyl)- (1-4)-2-methyl-(3,6-di-O-acetyl-1,2-dideoxy-alpha-D- glucopyrano)-[2,1-d]-2-oxazoline. This compound was phosphorylated by treatment with dibenzyl phosphate under strictly anhydrous conditions to give after catalytic hydrogenolysis, a peracetyl trisaccharide phosphate. This was coupled with P1-dolichyl P2-diphenyl diphosphate to give, after O-deacetylation, P1-dolichyl P2-[O- beta-D-mannopyranosyl- (1-4)-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)- (1-4)-2-acetamido-2-deoxy-alpha-D-glucopyranosyl] diphosphate, a trisaccharide "lipid intermediate". O-(2,3,4,6-Tetra-O-acetyl-alpha-D-mannopyranosyl)-(1-3)-O-(2,4,6-tri-O- acetyl-beta-D-mannopyranosyl)-(1-4)-O-(2-acetamido-3,6-di-O-acetyl-2- deoxy-beta- D-glucopyranosyl)- (1-4)-(2-acetamido-3,6-di-O-acetyl-2-deoxy-alpha-D-glucopyranosyl) phosphate, synthesized from O-alpha-D-mannopyranosyl-(1-3)-O-beta-D- mannopyranosyl-(1-4)-2-acetamido-2-deoxy-D-glucopyranose that had been isolated from mannosidosis urine was coupled with P1-dolichyl P2-diphenyl diphosphate to give, after O-deacetylation, P1-dolichyl P2-[O-alpha-D-mannopyranosyl- (1-3)-O-beta-D-mannopyranosyl-(1-4)-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-(1-4)-2-acetamido-2-deoxy-alpha-D-glucopyranosyl] diphosphate, a tetrasaccharide "lipid intermediate".

Preparation of a glycosyl donor suitable for synthesis of glycoprotein "core" oligosaccharides.

2-O-Acetyl-3-O-allyl-4-O-benzyl-6-O-tert-butyldiphenylsilyl-D-gluc opyranosyl chloride (14), a glycosyl donor suitable for synthesis of oligosaccharides corresponding to the N-glycoprotein saccharide "core", was synthesized by an efficient, six-stage route from 3,4,6-tri-O-acetyl-1,2-O-[1-(exo-ethoxy)ethylidene)-alpha-D-glucopyranos e. Silver triflate-promoted coupling of 14 with benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-alpha-D-glucopyranoside gave a protected beta-D-(1----4)-linked disaccharide in 38% yield, but a major side-reaction also occurred. When the tert-butyldiphenylsilyl group was quantitatively removed from 14 prior to the coupling reaction, and replaced afterwards, the yield in the glycosidation was increased to 55%, and major side-products were avoided.

The accumulation of oligosaccharides in tissues and body fluids of cats with alpha-mannosidosis.

Oligosaccharides were extracted from tissues and body fluids of five kittens with alpha-mannosidosis, three being from the same litter. The kittens were all of different ages at death and were compared to normal and heterozygote cats. The oligosaccharides were analyzed by high-pressure liquid chromatography after perbenzoylation and were identified by comparison with compounds of known structure. This provided a detailed picture of the distribution of oligosaccharides in each tissue, and a method for quantitation of the total oligosaccharides. With the exception of the youngest animal (death at day 2), the oligosaccharide elution profiles were broadly similar for all tissues and fluids, and were typical of feline alpha-mannosidosis. In contrast, concentrations of total oligosaccharides diverged widely from one source to another, from a high of 17.3 mumol/g to a low of 0.04 mumol/g. The results are interpreted in the context of glycoprotein catabolism.