Highly versatile solid phase synthesis of biofunctional 4-aryl-3,4-dihydropyrimidines using resin-bound isothiourea building blocks and multidirectional resin cleavage

A series of pharmacologically active, functionalized 4-aryl-3,4-dihydropyrimidine-5-carboxylates (DHPMs) are prepared by a versatile novel solid phase approach. In the key step, a polymer-bound thiouronium salt is condensed with unsaturated 1-ketoesters. The resulting polymer bound 1,4-dihydropyrimidines are cleaved from the resin employing multidirectional resin cleavage strategies.     

3'-Amino thymidine affinity matrix for the purification of herpes simplex virus thymidine kinase.

A simple procedure for preparation of an affinity resin with 3''-amino thymidine linked to the carboxyl residues on 6-amino-hexanoic agarose is described. We have used this column for a rapid and simple purification of the thymidine kinase encoded by the herpes simplex virus type 1 genome. This resin has two major advantages over the most widely use used resin made with thymidine-p-nitrophenyl phosphate: first it is easily obtainable, and second, it is not subject to destruction by phosphodiesterases. The two resins are very similar in behavior and the resin made with amino thymidine has allowed us to prepare large quantities of highly purified HSV TK for crystallization studies.     

Removal of boric acid and related compounds from solutions of carbohydrates with a boron-selective resin (IRA-743)

A boron-selective resin (IRA-743) was used to remove boric acid from synthetic solutions of carbohydrates. A commercially available polystyrene-divinylbenzene resin, which contains (covalently attached) 1-deoxy-1-methylamino-d-glucitol functional groups, absorbed > 11 mg of boric acid per mL of resin. Optimum conditions are described for use of this resin in a model system and in two practical applications: the quantitative removal of boric acid from a preparative-scale ( > 100 g) ketose-synthesis mixture and in the removal of boric acid from a solution of borohydride-reduced sugars (alditols) prior to acetylation and g.l.c. analysis.     

The occurrence of vertical resin canals in Keteleeria, with reference to its systematic position in Pinaceae

The variation in the number of resin canals in the secondary xylem of Keteleeria fortunei (A. Murray bis) Carrière is determined. At all levels from base to top of a tree, resin canals are absent rearer the pith, but they (sporadically) appear after a certain cambial age. In addition, the occurrence of resin canals in seven named species of Keteleeria was surveyed. It was confirmed that normal vertical resin canals do occur in the mature parts of secondary xylem, but that they may be absent in the juvenile parts of secondary xylem of stems, as well as in branches and in seedlings or saplings. Earlier investigations reporting the absence of resin canals altogether in this genus may have been misled by the use of younger (immature) specimens instead of mature stems of trees. The distribution pattern of resin canals found in Keteleeria is systematically significant because it supports a new system of classification of Pinaceae, in which Keteleeria is believed to have a close relationship with Nothotsuga.     

Maximizing the functional lifetime of Protein A resins

Protein A chromatography is currently the industry gold‐standard for monoclonal antibody and Fc‐fusion protein purification. The high cost of Protein A, however, makes resin lifetime and resin reuse an important factor for process economics. Typical resin lifetime studies performed in the industry usually examine the effect of resin re‐use on binding capacity, yield, and product quality without answering the fundamental question of what is causing the decrease in performance. A two part mechanistic study was conducted in an attempt to decouple the effect of the two possible factors (resin hydrolysis and/or degradation vs. resin fouling) on column performance over lifetime of the most commonly used alkali‐stable Protein A resins (MabSelect SuRe and MabSelect SuRe LX). The change in binding capacity as a function of sodium hydroxide concentration (rate of hydrolysis), temperature, and stabilizing additives was examined. Additionally, resin extraction studies and product cycling studies were conducted to determine cleaning effectiveness (resin fouling) of various cleaning strategies. Sodium hydroxide‐based cleaning solutions were shown to be more effective at preventing resin fouling. Conversely, cold temperature and the use of stabilizing additives in conjunction with sodium hydroxide were found to be beneficial in minimizing the rate of Protein A ligand hydrolysis. An effective and robust cleaning strategy is presented here to maximize resin lifetime and thereby the number of column cycles for future manufacturing processes. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:708–715, 2017     

Integrated antiherbivore, antidesiccant and ultraviolet screening properties of creosotebush resin

The surface leaf resin of creosotebush, Larrea spp. (Zygophyllaceae) exhibits antiherbivore, antidesiccant and UV screening properties. Composed mainly of monomeric phenolic aglycones, the resin absorbs most radiation of wavelength shorter than 410 nm, reduces the rate of evaporative water loss across cellulose membrane and exhibits tannin-like protein-complexing and digestibility-reducing properties. Leaf-chewing insects prefer mature leaves which have a lower resin content than young leaves. A phenoloxidaes system present in the leaves probably enhances the digestibility-reducing action of resin on in vitro proteolysis and growth-rates of Astroma quadrilobatum (Orthoptera: Proscopiidae) are negatively correlated with resin-phenoloxidase activity as measured by macerated leaf oxygen demand. Non-ideal behaviour of the resin is suggestive of cooperative action between components.     

Three-dimensional pore-scale modelling of dentinal infiltration

Dentine is the fundamental substrate of restorative dentistry, and its properties and characteristics are the key determinants of restorative processes. In contemporary restorative techniques, bonding to Dentine is created by the impregnation of the demineralised dentine by blends of resin monomers. In this paper, a numerical model of dentinal infiltration is proposed. The aim is to follow the resin front and to point out the optimal parameter set. The main tool is a level set technique to follow the evolving interface. It is coupled with the Navier–Stokes equation where capillary effect gives rise to the appearance of a new term in the variational approach than discretised by finite elements. Using an appropriate geometry representing demineralised dentine, the moving front is observed. First, a simulation of porosimetry test is achieved in order to validate the model. The two expected pore sizes are detected and the simulation also points out limitations of mercury intrusion porosimetry test in an educational way. Then a wetting fluid (representing the dental resin) is numerically infiltrated. In the dentinal porous network, capillarity is taken into account in our model by including a capillary term. A crucial conclusion is drawn from this study: resin application time by practitioners is sufficient if, in the infiltration process, the wetting phase is the resin.     

Assessment of the Acrylic Resin Technovit 7200 VLC for Studying the Gingival Mucosa by Light and Electron Microscopy

Technovit 7200 VLC is an acrylic resin formulated for embedding undecalcified hard tissues which are prepared for light microscopy according to a cutting-grinding technique. To employ this resin for embedding and cutting soft tissues by ultramicrotomy, we carried out a qualitative study on biopsies of canine gingival mucosa using light and transmission electron microscopy. For a critical evaluation of this resin, some biopsies were embedded in Agar 100, an epoxy resin widely used in morphological studies. At the light microscopic level the samples embedded in Technovit 7200 VLC showed good morphology and excellent toluidine blue staining of different cell types and extracellular matrix. At the ultrastrueturallevel, nuclei, cytoplasmic organelles, collagen fibrils and ground substance appeared well preserved and showed high electron density. The acrylic resin was stable under the electron beam and its degree of shrinkage appeared to be very low. We conclude that Technovit 7200 VLC can be employed for ultramicrotomy for both light and electron microscopic investigation of soft tissues.     

Assessment of the Acrylic Resin Technovit 7200 VLC for Studying the Gingival Mucosa by Light and Electron Microscopy

Technovit 7200 VLC is an acrylic resin formulated for embedding undecalcified hard tissues which are prepared for light microscopy according to a cutting-grinding technique. To employ this resin for embedding and cutting soft tissues by ultramicrotomy, we carried out a qualitative study on biopsies of canine gingival mucosa using light and transmission electron microscopy. For a critical evaluation of this resin, some biopsies were embedded in Agar 100, an epoxy resin widely used in morphological studies. At the light microscopic level the samples embedded in Technovit 7200 VLC showed good morphology and excellent toluidine blue staining of different cell types and extracellular matrix. At the ultrastrueturallevel, nuclei, cytoplasmic organelles, collagen fibrils and ground substance appeared well preserved and showed high electron density. The acrylic resin was stable under the electron beam and its degree of shrinkage appeared to be very low. We conclude that Technovit 7200 VLC can be employed for ultramicrotomy for both light and electron microscopic investigation of soft tissues.     

Evaluation of a novel methacrylate‐based protein a resin for the purification of immunoglobulins and Fc‐fusion proteins

Protein A affinity chromatography is a central part of most commercial monoclonal antibody and Fc‐fusion protein purification processes. In the last couple years an increasing number of new Protein A technologies have emerged. One of these new Protein A technologies consists of a novel, alkaline‐tolerant, Protein A ligand coupled to a macroporous polymethacrylate base matrix that has been optimized for immunoglobulin (Ig) G capture. The resin is interesting from a technology perspective because the particle size and pore distribution of the base beads are reported to have been optimized for high IgG binding and fast mass transfer, while the Protein A ligand has been engineered for enhanced alkaline tolerance. This resin was subjected to a number of technical studies including evaluating dynamic and static binding capacities, alkaline stability, Protein A leachate propensity, impurity clearance, and pressure–flow behavior. The results demonstrated similar static binding capacities as those achieved with industry standard agarose Protein A resins, but marginally lower dynamic binding capacities. Removal of impurities from the process stream, particularly host cell proteins, was molecule dependent, but in most instances matched the performance of the agarose resins. This resin was stable in 0.1 M NaOH for at least 100 h with little loss in binding capacity, with Protein A ligand leakage levels comparable to values for the agarose resins. Pressure–flow experiments in lab‐scale chromatography columns demonstrated minimal resin compression at typical manufacturing flow rates. Prediction of resin compression in manufacturing scale columns did not suggest any pressure limitations upon scale up. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1125–1136, 2014     

Effects of nitrogen fertilization on pine needle chemistry and sawfly performance

Summary Changes in needle nitrogen and resin acid concentrations in young Scots pine trees fertilized with ammonium nitrate were followed over 3 years. Sawfly larvae (Neodiprion sertifer) were reared on fertilized and control trees the year after fertilization. Both nitrogen and resin acid concentrations increased in fertilized trees. The fact that resin acid concentrations increased contradicts predictions of the carbon/nutrient balance hypothesis. We suggest that needle resin-acid concentrations are limited more by the size of the resin ducts than by the availability of substrate for resin acid synthesis, and that the formation of resin ducts is limited by the availability of nitrogen. A modification of the carbon/nutrient balance hypothesis, relating compartment formation to allelochemical synthesis, is discussed. Performance of sawfly larvae was not affected by fertilization treatment, probably because concentrations of nitrogen (positively affecting performance) and resin acids (adversely affecting performance) increased simultaneously in fertilized trees. Thus, the results of this study do not support the notion that fertilization increases the resistance of trees to needle-eating insects.     

Protein loading,elution, and resolution behavior in a novel device that integrates ultrafiltration and chromatographic separation

Hollow fiber membranes and chromatographic resin beads are commonly employed in a variety of bioseparation processes. A new class of integrated separation devices is being studied in which the shell side of a hollow fiber device is filled with adsorbents/chromatographic resin beads. Such devices and the corresponding separation methods integrate feed broth clarification by the microfiltration/ultrafiltration membrane with bioproduct purification by the shell-side resin beads either as an adsorbent or as beads in elution chromatography. A mathematical model has been developed for the prediction of the chromatographic behavior of such an integrated device. Simulations have been done to study the effects of axial dispersion, feed flow rate, water permeation rate, fiber packing density, and void fraction. Numerical solutions were obtained by solving the governing equations. This model can reasonably describe the concentration profiles as well as the breakthrough and elution behaviors in the integrated device.     

A spectrophotometric resin competition procedure to determine dissociation constants for metal ion-nucleotide complexes

The resin competition method for determining the dissociation constants of metal ion-nucleotide complexes was modified to take into account the fact that some metal nucleotide complexes are anionic and thus, like the free nucleotide, will bind to the resin. A simple, rapid spectrophotometric titration procedure is given for the determination of both the metal ion-nucleotide complex dissociation constant and the ratio of the affinities of the nucleotide and its complex for the resin.     

General method for rapid synthesis of multicomponent peptide mixtures

A method is suggested for the synthesis of multicomponent peptide mixtures. The method is a solid phase synthesis modified in order to give a closely equimolar mixture of peptides with predetermined sequences. The main point of modification is that before every coupling cycle the resin is divided into equal parts and each portion is coupled with a different amino acid. Then the portion are mixed and before the next coupling cycle the resin is again distributed into equal portions. The method is illustrated by the synthesis of a mixture of 27 tetrapeptides and that of 180 pentapeptides.     

In situ RNA hybridization using Technovit resin in Arabidopsis thaliana

A protocol is described for RNA in situ hybridization using thin sections prepared by Technovit resin. Technovit is a widely used resin for histological examinations. Since it does not require time-consuming processes such as removal of the resin and can be performed without high temperature treatment, a high resolution of sections could be possible compared to other resins and paraffin. Thin sections (approximately 4 m) were made from inflorescences of Arabidopsis thaliana embedded in Technovit 8100 resin, and in situ hybridization was performed using the protocol described in this article. Hybridization signals were observed using LEAFY and other genes as probes, showing that this resin can be used for in situ analysis. In our experiments, the most important factor for a successful in situ hybridization pattern was to optimize the RNase A concentration after hybridization. We routinely used RNase A at a concentration of 2–5 ng/ml, a concentration much lower than that used for paraffin embedding method. Thus, the use of the Technovit resin for plant tissue embedding results in a faster protocol and greater quality than allowed by paraffin sections.     

Simultaneous Demonstration of Lipids and Starch in Plant Tissues

A rapid and easy technique for the simultaneous demonstration of lipids and starch in the same histological section is described. Tissues are prepared by the clamical fixing and Araldite in embedding techniques of election microscopy. Semithin sections are directly stained for 1 hour at 60C with saturated Sudan black B in 70% ethanol without removing the embedding resin. Lipids stain black; stain is shown as white grains contrasting with the blue-grey embedding resin.     

Observations on the use of a cation exchange resin for the preparation of metal-depleted media for lymphocyte culture

A chelating resin specific for divalent cations (Chelex) was used to prepare metal-depleted media for lymphocyte culture. A batch procedure (resin in pH 7.4 phosphate buffer/specimen, 1:1) removed 70-80% of iron, 77-87% of copper and 88-98% of zinc, calcium and magnesium. At variance with other reports, when a resin/specimen ratio of 1:4 was used, iron chelation decreased to 40%, whereas other cation chelation remained unchanged. Best chelation for iron and calcium was obtained at pH 5-6.4; for copper, zinc and magnesium, at pH 7.4-8.0. During the procedure protein content decreased by 8-10%; arginine and lysine by 80%; asparagine, cystine, tyrosine and phenylalanine by 60%, other amino acids by 35%. These new data suggest that cation-depleted media prepared with Chelex may be used to study the effects of cations on lymphocytes in culture, provided that the most appropriate pH and resin/specimen ratio are selected and adequate amino acid replacement is performed. Results on normal human lymphocytes are reported.     

Properties of flaky affinity resin with co-continuous structure

A new type of flaky affinity resin for capture of the target proteins was prepared to discuss its properties compared with those of a particulate affinity resin. The resin prepared had totally co-continuous structure (monolith) and was utilized in the shape of flake. The concentration of surface amino groups for immobilization of ligand was determined to be 22.3 micromol/ml. Immobilizations of ligand such as Sulfonamide, Ketoprofen, Captopril, or Methotrexate (MTX) on the affinity resin were quantitatively proceeded to afford fully covered (100%) affinity resins. Control of the immobilization rate of affinity resin using Sulfonamide or Ketoprofen was successfully achieved with the calculated immobilization rate. The flaked shape of affinity resin (100-400 microm) presumably simplified affinity experimental procedures and the affinity resin immobilizing Sulfonamide effectively captured one of the target proteins, CAII, without non-specifically bound proteins. The observed properties of the flaky affinity resin as well as ease in handling are really useful for capture of the target proteins of possible rare ligands.     

Simultaneous demonstration of lipids and starch in plant tissues.

A rapid and easy technique for the simultaneous demonstration of lipids and starch in the same histological section is described. Tissues are prepared by the classical fixing and Araldite M embedding techniques of electron microscopy. Semithin sections are directly stained for 1 hour at 60C with saturated Sudan black B in 70% ethanol without removing the embedding resin. Lipids stain black; starch is shown as white grains contrasting with the blue-grey embedding resin.     

Biotransformation of cystothiazole A, a myxobacterial antibiotic, into novel derivatives by the mother producer, Cystobacter fuscus

The bioconversion of the myxobacterial antibiotic, cystothiazole A, by the antibiotic producer, Cystobacter fuscus, was investigated. In our previous study, an adsorbent resin was added to the fermentation mixture to achieve high productivity of cystothiazole A, the major and most active component. On the other hand, a relative increase in the metabolic derivatives of cystothiazole A was observed when cultured without the resin. Furthermore, when cystothiazole A was externally added to the culture of C. fuscus without the resin, cystothiazole A was rapidly metabolized by the culture to a number of polar metabolic derivatives, among them being novel ones. The identification and structural elucidation of the known and novel derivatives were performed by spectroscopic analyses. Based on the time-dependent production profile and chemical structures of these derivatives, pathways for the conversion of cystothiazole A to the more polar derivatives of this antibiotic by C. fuscus are proposed.