Back Cover: Protein secondary structure analysis of dried blood serum using infrared spectroscopy to identify markers for colitis screening (J. Biophotonics 3/2018)

Protein secondary structural alteration in the serum sample as induced by colitis has been demonstrated via the spectral fitting. Using DSS mouse models of acute colitis and IL10‐/‐ for chronic colitis, a significant difference in the integral ratio of Gaussian energy bands representing α‐helix and β‐pleated sheet structures were obtained. Further details can be found in the article by Jitto Titus et al. ( e201700057 ).

     

Minimally invasive screening for colitis using attenuated total internal reflectance fourier transform infrared spectroscopy

This article describes a rapid, simple and cost‐effective technique that could lead to a screening method for colitis without the need for biopsies or in vivo measurements. This screening technique includes the testing of serum using Attenuated Total Reflectance Fourier Transform Infrared (ATR‐FTIR) spectroscopy for the colitis‐induced increased presence of mannose. Chronic (Interleukin 10 knockout) and acute (Dextran Sodium Sulphate‐induced) models for colitis are tested using the ATR‐FTIR technique. Arthritis (Collagen Antibody Induced Arthritis) and metabolic syndrome (Toll like receptor 5 knockout) models are also tested as controls. The marker identified as mannose uniquely screens and distinguishes the colitic from the non‐colitic samples and the controls. The reference or the baseline spectrum could be the pooled and averaged spectra of non‐colitic samples or the subject's previous sample spectrum. This shows the potential of having individualized route maps of disease status, leading to personalized diagnosis and drug management.

     

Isolation and identification of mannose‐binding proteins and estimation of their abundance in sera from hepatocellular carcinoma patients

The interaction of glycan‐binding proteins (GBPs) and glycans plays a significant biological role that ranges from cell–cell recognition to cell trafficking, and glycoprotein targeting. The anomalies of GBPs related to the types and/or quantities were not clearly known in cancer incidence. It is imperative to identify and annotate the GBPs related with the canceration. Here the mannose‐binding proteins (MBPs) from the clinical sera were isolated and identified by the mannose‐magnetic particle conjugates and the high‐accuracy MS analysis. Seventy‐five MBPs from normal donors’ sera and 79 MBPs from hepatocellular carcinoma patients’ sera were identified and annotated. By using the stringent criteria of exponentially modified protein abundance index (emPAI) quantification, 12 MBPs were estimated to be significantly upregulated (emPAI ratio > 4) and nine MBPs were estimated to be significantly downregulated (emPAI ratio < 0.25) in the hepatocellular carcinoma sera. Real‐time quantitative PCR, Western blotting, and protein microarrays were also used to confirm the altered MBPs expression level and the specific binding between the isolated MBPs and mannose. The sequence recognition motifs and structure preference of the isolated MBPs were characterized. The functional enrichment analysis revealed that over 57% of the isolated MBPs were binding protein and the upregulated MBPs were involved in cell death, tumor progression, and macromolecular complex remodeling.     

Dolichyl phosphate linked sugars as intermediates in the synthesis of yeast mannoproteins: an in vivo study

Freshly prepared protoplasts of Saccharomyces cerevisiae X 2180 incorporate [³H]mannose and [¹⁴C]glucose for about 30 min into glycolipids and mannoproteins. Among the radioactive glycolipids formed dolichyl phosphate mannose, dolichyl phosphate glucose and dolichyl pyrophosphate oligosaccharides have been identified. The oligosaccharides released by weak acid from the dolichyl pyrophosphate were treated with endo-N-acetylglucosaminidase H and separated by gel filtration on Bio-Gel P-4. The largest oligosaccharide obtained corresponded exactly in size to Glc₃Man₉GlcNAc₁ the compound formed also in animal tissues. Other oligosaccharides released from dolichyl pyrophosphate in addition to the glucose containing ones were mainly Man₉GlcNAc₁ and Man₈GlcNAc₁. No mannosyl oligosaccharide corresponding in size to the total inner core region found in native mannoproteins could be detected in a lipid-bound form.The radioactive dolichyl pyrophosphate oligosaccharides were formed transiently; after 40 min only about 40% of the maximal radioactivity was observed in this fraction. In the presence of cycloheximide this decrease did not take place.It is concluded that the dolichol pathway of N-glycosylation of glycoproteins in yeast cells is very similar, if not identical, to the reaction sequence worked out for animal cells.Dedicated to Professor Dr. Otto Kandler on his 60th birthday     

Evidence against the presence of fibres or chemically distinct layers in the cell wall ofSaccharomyces

The gross chemical composition of material extracted from yeast cell walls with various solvents or enzymes was studied. Attempts were made to locate these materials in situ by comparing electron micrographs of negatively stained and sectioned cell walls with those of the residues of the extraction procedures. There are at least two chemically distinct species of carbohydrate polymers which can be extracted with strong alkali: one yielding mainly mannose and some amino acid on hydrolysis and the other yielding mannose, glucose and amino acid. The alkali-insoluble material also yielded glucose, mannose and amino acid on hydrolysis but the glucose/mannose ratio was much higher. It was shown that none of these polymers constituted a physically distinct layer in the yeast cell wall. However, there does seem to be a region at the outer surface with distinctive properties. This is not fibrillar in nature and after extraction with ethylene diamine forms a double-layered structure. Materials which react with KMnO₄ to produce an electron-dense material are located throughout the wall but tend to be concentrated in the outer and inner regions. Procedures which remove this material also remove up to 80% of the mannose, 40% of the glucose and 35 % of the protein of the original wall material. It was shown that fibres do not constitute a major fraction of the normal cell wall, except possibly in the region of the bud scars but may be produced fairly readily by certain specific treatments. The classical view of the yeast cell wall with the structural integrity being maintained by a fibrous network of 1–3, 1–6 linked glucose residues is challenged and evidence to support an alternative view is presented.The results in wthis paper were presented to the University of Manchester Institute of Science and Technology by JKB in a Thesis for the degree of M.Sc. (Bowden, 1966). The authors are indebted to Miss C. Backhouse and Miss B. Murphy for help in preparing the electron micrographs.     

Phosphomannomutase activity in wild-type and alginate-producing strains ofPseudomonas aeruginosa

Phosphomannomutase (PMM) activity was detected in the soluble cytoplasmic fraction of crude extracts of both mucoid (alginate-producing) and nonmucoid strains ofPseudomonas aeruginosa. The enzyme activity was concentrated and partially purified from cell extracts of mucoid strain V388 by precipitation with ammonium sulfate and by molecular exclusion chromatography. These preparations catalyzed the conversion of mannose 1-phosphate to mannose 6-phosphate in a coupled assay system that contained commercial phosphomannoisomerase, phosphoglucoisomerase, and glucose 6-phosphate dehydrogenase. Catalytic activity in this system was strictly dependent on the presence of glucose 1,6-diphosphate (apparent Km, 150 M) and exhibited a pH optimum of around 9 in Bicine-NaOH buffer. PMM exhibited an apparent Km of 60 M for mannose 1-phosphate, but concentrations greater than 150 M caused significant inhibition. Specific activities of PMM were consistently higher in the soluble fractions of mucoid strains (1.2–3.6 nmol/min/mg protein) than of nonmucoid strains (0.2–0.6 nmol/min/mg protein).     

The Metabolic Origins of Mannose in Glycoproteins

Mannose in N-glycans is derived from glucose through phosphomannose isomerase (MPI, Fru-6-P ↔ Man-6-P) whose deficiency causes a congenital disorder of glycosylation (CDG)-Ib (MPI-CDG). Mannose supplements improve patients'' symptoms because exogenous mannose can also directly contribute to N-glycan synthesis through Man-6-P. However, the quantitative contributions of these and other potential pathways to glycosylation are still unknown. We developed a sensitive GC-MS-based method using [1,2-¹³C]glucose and [4-¹³C]mannose to measure their contribution to N-glycans synthesized under physiological conditions (5 mm glucose and 50 μm mannose). Mannose directly provides ∼10–45% of the mannose found in N-glycans, showing up to a 100-fold preference for mannose over exogenous glucose based on their exogenous concentrations. Normal human fibroblasts normally derive 25–30% of their mannose directly from exogenous mannose, whereas MPI-deficient CDG fibroblasts with reduced glucose flux secure 80% of their mannose directly. Thus, both MPI activity and exogenous mannose concentration determine the metabolic flux into the N-glycosylation pathway. Using various stable isotopes, we found that gluconeogenesis, glycogen, and mannose salvaged from glycoprotein degradation do not contribute mannose to N-glycans in fibroblasts under physiological conditions. This quantitative assessment of mannose contribution and its metabolic fate provides information that can help bolster therapeutic strategies for treating glycosylation disorders with exogenous mannose.     

Hyperspectral scanning laser optical tomography

In order to study physical relationships within tissue volumes or even organism‐level systems, the spatial distribution of multiple fluorescent markers needs to be resolved efficiently in three dimensions. Here, rather than acquiring discrete spectral images sequentially using multiple emission filters, a hyperspectral scanning laser optical tomography system is developed to obtain hyperspectral volumetric data sets with 2‐nm spectral resolution of optically transparent mesoscopic (millimeter‐centimeter) specimens. This is achieved by acquiring a series of point‐scanning hyperspectral extended depth of field images at different angles and subsequently tomographically reconstructing the 3D intensity distribution for each wavelength. This technique is demonstrated to provide robust measurements via the comparison of spectral and intensity profiles of fluorescent bead phantoms. Due to its enhanced spectral resolving ability, this technique is also demonstrated to resolve largely overlapping fluorophores, as demonstrated by the 3D fluorescence hyperspectral reconstruction of a dual‐labeled mouse thymus gland sample and the ability to distinguish tumorous and normal tissues of an unlabeled mouse intestine sample.      

Dual‐polarization Raman spectral imaging to extract overlapping molecular fingerprints of living cells

Raman spectral imaging is gaining more and more attention in biological studies because of its label‐free characteristic. However, the discrimination of overlapping chemical contrasts has been a major challenge. In this study, we introduce an optical method to simultaneously obtain two orthogonally polarized Raman images from a single scan of the sample. We demonstrate how this technique can improve the quality and quantity of the hyperspectral Raman dataset and how the technique is expected to further extend the horizons of Raman spectral imaging in biological studies by providing more detailed chemical information.

The dual‐polarization Raman images of a HeLa cell.     

Rapid intra‐operative diagnosis of kidney cancer by attenuated total reflection infrared spectroscopy of tissue smears

Herein, a technique to analyze air‐dried kidney tissue impression smears by means of attenuated total reflection infrared (ATR‐IR) spectroscopy is presented. Spectral tumor markers—absorption bands of glycogen—are identified in the ATR‐IR spectra of the kidney tissue smear samples. Thin kidney tissue cryo‐sections currently used for IR spectroscopic analysis lack such spectral markers as the sample preparation causes irreversible molecular changes in the tissue. In particular, freeze‐thaw cycle results in degradation of the glycogen and reduction or complete dissolution of its content. Supervised spectral classification was applied to the recorded spectra of the smears and the test spectra were classified with a high accuracy of 92% for normal tissue and 94% for tumor tissue, respectively. For further development, we propose that combination of the method with optical fiber ATR probes could potentially be used for rapid real‐time intra‐operative tissue analysis without interfering with either the established protocols of pathological examination or the ordinary workflow of operating surgeon. Such approach could ensure easier transition of the method to clinical applications where it may complement the results of gold standard histopathology examination and aid in more precise resection of kidney tumors.      

Mutation screening by a combination of biotin-SSCP and direct sequencing

We have developed a mutation detection strategy that combines single strand conformational polymorphism (SSCP) analysis of one strand of a double-stranded amplification product with direct sequencing of the other. Using this strategy, which we find economical of both time and resources, we have identified a G to A transition, which substitutes a serine for glycine residue at position 862 in the major helix of the 1 chain of Type I collagen. We use this mutation, which causes a lethal form of osteogenesis imperfecta, to illustrate the technique.     

Developing Raman spectroscopy as a diagnostic tool for label‐free antigen detection

For several decades, a multitude of studies have documented the ability of Raman spectroscopy (RS) to differentiate between tissue types and identify pathological changes to tissues in a range of diseases. Furthermore, spectroscopists have illustrated that the technique is capable of detecting disease‐specific alterations to tissue before morphological changes become apparent to the pathologist. This study draws comparisons between the information that is obtainable using RS alongside immunohistochemistry (IHC), since histological examination is the current GOLD standard for diagnosing a wide range of diseases. Here, Raman spectral maps were generated using formalin‐fixed, paraffin‐embedded colonic tissue sections from healthy patients and spectral signatures from principal components analysis (PCA) were compared with several IHC markers to confirm the validity of their localizations. PCA loadings identified a number of signatures that could be assigned to muscle, DNA and mucin glycoproteins and their distributions were confirmed with antibodies raised against anti‐Desmin, anti‐Ki67 and anti‐MUC2, respectively. The comparison confirms that there is excellent correlation between RS and the IHC markers used, demonstrating that the technique is capable of detecting compositional changes in tissue in a label‐free manner, eliminating the need for antibodies.      

Production of an extracellular polysaccharide byAgrobacterium sp DS3 NRRL B-14297 isolated from soil

A bacterium isolated from soil and identified asAgrobacterium sp produced a water-soluble extracellular polysaccharide capable of producing highly viscous solutions. Gas chromatographic analysis revealed a sugar composition of glucose, galactose and mannose in the molar ratio of 7.52.41, together with 3.7% (w/w) pyruvic acid. Methylation analyses showed the presence of (13)-, (14)- and (16)-linked glucose, (13)- and (14, 16)-linked galactose and a small portion of (13)-linked mannose residues. Succinic acid was not present. The molecular weight of the polysaccharide was estimated by light scattering to be 2×10⁶ Da. The viscosity of solutions containing the polysaccharide remained constant from pH 3 to 11, and decreased by 50% when heated from 5 to 55°C. Maximum yield of the polysaccharide, 20 g L^–1, was reached in 48 h at 30°C incubation.     

Characteristics of a novel high viscosity polysaccharide,methylan, produced byMethylobacterium organophilum

Summary A new anionic high molecular weight polysaccharide, Methylan, was produced byMethylobacterium organophilum from methanol as a sole source of carbon and energy under the specific culture conditions. By GPC and light scattering, the molecular weight was determined to be 2–4×10⁶ dalton and the distribution of molecular weight was very homogeneous. Methylan was composed of carbohydrate (80%), uronic acid (12%), protein (6%) and pyruvic acid (5%). The sugar composition of Methylan was identified as glucose, galactose and mannose with the approximate molar ratio of 232. Methylan solution showed a pseudoplastic non-Newtonian fluid property at the concentration above 0.05%. At the concentration of 1% Methylan solution, the consistency index was 18,000 centipoise which was almost 10 times higher than that of Xanthan and the flow behavior index was 0.15.     

Restriction fragment length polymorphism (RFLP) analyses of plants produced by in vitro anther culture of Solanum chacoense Bitt

Summary Green mesophyll protoplasts of the dihaploid potato line 1982 (Solanum tuberosum L.) were fused with herbicide-bleached mesophyll protoplasts of the dihaploid potato line 679 using a polyethylene glycol protocol. Heterokaryons were identified under a fluorescence microscope using the dual fluorescence of carboxyfluorescein-stained, herbicide-bleached protoplasts and the autofluorescence of green mesophyll protoplasts. About 20% of the protoplasts survived the fusion treatment, and the fusion frequency was 3%–4%. Unfused and fused protoplasts were mass cultured for 6 weeks after which vigorously growing calli were selected and transferred to shoot regeneration medium. Somatic hybrids were identified by a combination of five isozyme markers, and the ploidy level was determined by flow cytometry. Out of 15 calli that regenerated shoots, 6 plants derived from 2 different calli were identified as hexaploid somatic hybrids, while one morphologically deviant plant from a third callus was identified as a mixoploid that had lost some enzyme markers after 4 months of culturing.     

Re-sorption of organic components by roots of Zea mays L. and its consequences in the rhizosphere

The re-sorption of carbon compounds from the rhizosphere was investigated using ¹⁴C-labelled glucose, mannose and citric acid. Uptake in roots of 5-day-old, intact Zea mays plants in sterile solution culture was determined over a period of 48 hours. Under optimal growth conditions significant re-absorption of glucose and mannose occurred with the uptake rates being 70.5 and 40.2 g compound g^-1 root DW h^-1, respectively. For glucose and mannose approximately 25% of the ¹⁴C label taken up by the root was recovered inside the plant as low-MW compounds and 33% polymerized into high MW compounds. 42% was respired as ¹⁴C-CO₂. Citric acid by comparison showed little accumulation within plant tissues (11.4%) with most being respired and recovered as ¹⁴C-CO₂ in KOH traps (88%). The uptake rate for citric acid was 34.8 g g^-1 root DW h^-1. Over the 48-hour period a net efflux (i.e. exudation) of labelled plus unlabelled C was observed at a rate of 608 g C g^-1 root DW h^-1 (equivalent to 1520 g glucose/mannose). Of the C released as root exudates, a minimum estimate of the amount of C taken back into the plant was therefore 9.5%. The two main C fluxes within the rhizosphere, namely release of C by the root and uptake by the microorganisms, have been well documented in recent years. It is now apparent however that a third flux term, re-sorption of C by roots, can also be identified. This may play an important but previously overlooked role within the rhizosphere, and further work is needed to determine its significance.A comparison between exudate release in static (permitting accumulation of C) and flowing culture (C removed as it is released) was also made with the respective rates being 15.36 and 45.18 mg C g^-1 root DW in 2 days. The relative important of re-sorption in natural environments and laboratory experiments is discussed.     

Reduced Na+/K+ ATPase transport activity,resting membrane potential,and bradykinin-stimulated phosphatidylinositol synthesis by polyol accumulation in cultured neuroblastoma cells

In these studies we examined the effect of polyol accumulation on neural cellmyo-inositol metabolism and properties. Neuroblastoma cells were cultured for two weeks in media containing 30 mM glucose, fructose, galactose or mannose with or without 0.4 mM sorbinil or 250 Mmyo-inositol. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a decrease inmyo- inositol content and myo-[2-³H]inositol accumulation and incorporation into phosphoinositides compared to cells cultured in unsupplemented medium or medium containing 30 mM fructose as an osmotic control. These monosaccharides each caused an increase in intracellular polyol levels with galactitol > sorbitol = mannitol accumulation. Chronic exposure of neuroblastoma cells to media containing 30 mM glucose, galactose, or mannose caused a significant decrease in Na⁺/K⁺ ATPase transport activity, resting membrane potential, and bradykinin-stimulated³²P incorporation into phosphatidylinositol compared to cells cultured in medium containing 30 mM fructose. In contrast, basal incorporation of³²P into phosphatidylinositol or basal and bradykinin-stimulated³²P incorporation into phosphatidylinositol 4,5-bisphosphate were not effected. Each of these cellular functions as well asmyo-inositol metabolism and content and polyol levels remained near control values when 0.4 mM sorbinil, an aldose reductase inhibitor, was added to the glucose, galactose, or mannose supplemented media.myo-Inositol metabolism and content and bradykinin-stimulated phosphatidylinositol synthesis were also maintained when media containing 30 mM glucose, galactose, or mannose was supplemented with 250 Mmyo-inositol. The results suggest that polyol accumulation induces defects in neural cellmyo-inositol metabolism and certain cell functions which could, if they occurred in vivo, contribute to the pathological defects observed in diabetic neuropathy.     

Comparing the sugar profiles and primary structures of alkali-extracted water-soluble polysaccharides in cell wall between the yeast and mycelial phases from <Emphasis Type="Italic">Tremella fuciformis</Emphasis>

To gain insights into dimorphism, cell wall polysaccharides from Tremella fuciformis strains were obtained from alkali-extracted water-soluble fractions PTF-M38 (from the mycelial form), PTF-Y3 and PTF-Y8 (from the yeast form) of T. fuciformis strains were used to gain some insights into dimorphism study. Their chemical properties and structural features were investigated using gel permeation chromatography, gas chromatography, UV and IR spectrophotometry and Congo red binding reactions. The results indicated that the backbones of PTF-M38, PTF-Y3 and PTF-Y8 were configured with α-linkages with average molecular weights of 1.24, 1.08, and 1.19 kDa, respectively. PTF-M38 was mainly composed of xylose, mannose, glucose, and galactose in a ratio of 1:1.47:0.48:0.34, while PTF-Y3 and PTF-Y8 were mainly composed of xylose, mannose and glucose in a ratio of 1:1.65:4.06 and 1:1.21:0.44, respectively. The sugar profiles of PTF-M38, PTF-Y3 and PTF-Y8 were also established for further comparison. These profiles showed that all three polysaccharides contained the same sugars but in different ratios, and the carbon sources (xylose, mannose, glucose, and galactose) affected the sugar ratios within the polysaccharides.     

Towards an integrated linkage map of common bean 2. Development of an RFLP-based linkage map

Summary A restriction fragment length polymorphism (RFLP)-based linkage map for common bean (Phaseolus vulgaris L.) covering 827 centiMorgans (cM) was developed based on a F₂ mapping population derived from a cross between BAT93 and Jalo EEP558. The parental genotypes were chosen because they exhibited differences in evolutionary origin, allozymes, phaseolin type, and for several agronomic traits. The segregation of 152 markers was analyzed, including 115 RFLP loci, 7 isozyme loci, 8 random amplified polymorphic DNA (RAPD) marker loci, and 19 loci corresponding to 15 clones of known genes, 1 virus resistance gene, 1 flower color gene, and 1 seed color pattern gene. Using MAPMAKER and LINKAGE-1, we were able to assign 143 markers to 15 linkage groups, whereas 9 markers remained unassigned. The average interval between markers was 6.5 cM; only one interval was larger than 30 cM. A small fraction (9%) of the markers deviated significantly from the expected Mendelian ratios (121 or 31) and mapped into four clusters. Probes of known genes belonged to three categories: seed proteins, pathogen response genes, and Rhizobium response genes. Within each category, sequences homologous to the various probes were unlinked. The I gene for bean common mosaic virus resistance is the first disease resistance gene to be located on the common bean genetic linkage map.     

Release of glucose-mediated catabolite repression due to a defect in the membrane fraction of phosphoenolpyruvate: mannose phosphotransferase system in Pediococcus halophilus

A spontaneous mutant 9R-4 resistant to 2-deoxyglucose (2DG) was derived from a wild-type strain Pediococcus halophilus I-13. Phosphoenolpyruvate (PEP)-dependent glucose-6-phosphate formation by the permeabilized 9R-4 cells was < 5% of that observed with the parent I-13. In vitro complementation of PEP-dependent 2DG-6-phosphate formation was assayed with combination of the cytoplasmic and membrane fractions prepared from the I-13 and the mutants (9R-4, and X-160 isolated from nature), which were defective in PEP: mannose phosphotransferase system (man:PTS). The defects in man:PTS of both the strain 9R-4 and X-160 were restricted to the membrane fraction (e.g. EII^man), not to the cytoplasmic one. Kinetic studies on the glucose transport with intact cells and iodoacetate-treated cells also supported the presence of two distinct transport systems in this bacterium as follows: (i) The wild-type I-13 possessed a high-affinity man:PTS (K _m=11 M) and a low-affinity proton motive force driven glucose permease (GP) (K _m=170 M). (ii) Both 9R-4 and X-160 had only the low-affinity system (K _m=181 M for 9R-4, 278 M for X-160). In conclusion, a 2DG-induced selective defect in the membrane component (EII^man) of the man:PTS could partially release glucose-mediated catabolite repression but not frutose-mediated catabolite repression in soy pediococci.Abbreviations GCR glucose-mediated catabolite repression - FCR fructose-mediated catabolite repression - PEP phosphoenolpyruvate - man:PTS phosphoenolpyruvate:mannose phosphotransferase system - glc:PTS phosphoenolpyruvate:glucose phosphotransferase system - GP glucose permease - CCCP carbonylcyanide mchlorophenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide - P proton motive force - G-6-P glucose-6-phosphate - 2DG 2-deoxyglucose - IAA iodoacetic acid - EII^man enzyme II component of man:PTS - EIII^man enzyme III component of man:PTS - EII^glc enzyme II component of glc:PTS - EIII^glc enzyme III component of glc:PTS