Influence of PEG endgroup and molecular weight on its reactivity for lipase-catalyzed polyester synthesis

Polycondensations were performed at 70 degrees C in bulk using physically immobilized lipase B from Candida antarctica (CAL-B) as catalyst. Study of copolymerizations between sebacic acid and PEG diols of differing Mn values (200, 400, 600, 1000, 2000, and 10 000) showed that PEG 400 and 600 were most reactive (DP(avg) up to about 6). Increasing the PEG diol chain length from 600 to 1000, 2000, and 10 000 resulted in large decreases in copolymer DP(avg) values. PEG200 diacids (i.e., HOOC-(CH2)x-O-(CH2CH2O)n-(CH2)x-COOH) were successfully synthesized where x was 1, 4, 5, 7, 9, and 11. Study of copolymerizations of these diacids with 1,8-octanediol showed that, by introduction of a five-carbon methylene spacer (x = 5), remarkable increases in the reactivity of PEG200 diacids were achieved. In addition, introduction of this spacer was also effective for increasing the reactivity of PEG diacids of higher molecular weight (i.e., PEG400, 600, and 1000). This work verified the hypothesis that, by conversion of PEG chain ends to structures more closely resembling fatty acids, modified PEG building blocks are obtained that are better recognized as substrates by CAL-B during condensation reactions.     

New aspects of bacterial ultrastructure as revealed by modern acrylics for electron microscopy

Modern acrylics can be used over a wide temperature range (+60 degrees C to -80 degrees C) for infiltration, embedding, and polymerization. They can be used in procedures involving chemical fixation or rapid freezing. This flexibility allows for studies to be carried out upon the effects that different parameters involved in preparing biological tissue for microscopy have upon structure and retention of immunoreactivity. With most preparative methods contributions have been made to our knowledge on bacterial structure in gram-negative and gram-positive cells. The future should lie in integrating the advantages of the various methods for the purpose of advancing our understanding of bacterial structure/function.     

Polyethylene glycol embedded tissue sections for immunoelectronmicroscopy

Summary Several methods for tissue embedding in polyethylene glycol (PEG) were compared with regard to their applicability for pre-embedding immunoelectronmicroscopy. Existing embedding procedures gave unsatisfactory results and therefore a modified procedure was developed. This method, consisting of very brief tissue infiltration with PEG 1500, to which 3% water is added, allowed adequate tissue sectioning. Using these sections for preembedding immunoelectronmicroscopical localisation of glucagon in bovine pancreatic islets adequate ultrastructural morphology was obtained in combination with excellent preservation of peptide hormone immunoreactivity.Supported in part by grant no. PAL 52-77 of the Queen Qilhelmina Cancer Foundation     

Glycol Methacrylate in Light Microscopy a Routine Method for Embedding and Sectioning Animal Tissues

Glycol methacrylate (GMA), a water and ethanol miscible plastic, was introduced to histology as an embedding medium for electron microscopy. This medium may be made soft enough for cutting thick sections for routine light microscopy by altering its composition. A procedure for the infiltration, polymerization, and sectioning of animal tissues in GMA for light microscopy is presented which is no more complex than paraffin techniques and which has a number of advantages: (I) The GMA medium is compatible with both aqueous fixatives (formaldehyde, glutaraldehyde, Bouin's, and Zenker's) and non-aqueous fixatixes (Carnoy's, Newcomer's, ethanol, and acetone). (2) Undue solvent extraction of the tissue is avoided because adequate dehydration occurs during infiltration of the embedding medium. Separate dehydration and clearing of the tissue prior to embedding is eliminated. (3) When polymerized, the supporting matrix is firm enough that hard and soft tissues adjacent to one another may be sectioned without distortion. (4) Thermal artifact is reduced to a minimum during polymerization because the temperature of the tissue may be maintained at 0-4 C from fixation through ultraviolet light polymerization of the embedding medium. (5) Shrinkage during polymerization of the embedding medium is minimized by prepolymerization of the medium before use. (6) Sections may be easily cut using conventional steel knives and rotary microtomes at a thickness of 0.5 to 3.0 microns, thus improving resolution compared with routinely thicker paraffin sections. (7) The polymerized GMA medium is porous enough so that staining, auto radiography, and other histological procedure are done without removal of the embedding medium from the sections. A list of these stains and related procedures are included. (8) Enzyme digestion of ultra thin sections of tissue embedded in GMA is common in electron microscopic cyto chemistry. me same digestion techniques appear compatible with the thicker seaions used in light microscopy.     

Effects of peptide molecular mass and PEG chain length on the vasoreactivity of VIP and PACAP(1-38) in pegylated phospholipid micelles

Bioactive properties of certain amphipathic peptides are amplified when self-associated with sterically stabilized micelles (SSM) composed of polyethylene glycol (PEG)-conjugated phospholipids. The purpose of this study was to determine the effects of amphipathic peptide molecular mass and PEG chain length on vasoreactivity evoked by vasoactive intestinal peptide (VIP), a 28-amino acid neuropeptide, and pituitary adenylate cyclase-activating peptide(1-38) (PACAP(1-38)), a 38-amino acid neuropeptide, associated with PEGylated phospholipid micelles in vivo. Both peptides were incubated for 2 h with SSM composed of PEG with molecular mass of 2000 or 5000 grafted onto distearoyl-phosphatidylethanolamine (DSPE-PEG2000 or DSPE-PEG5000) before use. We found that regardless of peptide molecular mass, PEG chain length had no significant effects on peptide-SSM interactions. Using intravital microscopy, VIP associated with DSPE-PEG5000 SSM or DSPE-PEG2000 SSM incubated at 25 degrees C evoked similar vasodilation in the intact hamster cheek pouch microcirculation. Likewise, PACAP(1-38)-induced vasodilation was PEG chain length-independent. However, SSM-associated PACAP(1-38) evoked significantly smaller vasodilation than that evoked by SSM-associated VIP (P < 0.05) at 25 degrees C. When the incubation temperature was increased to 37 degrees C, SSM-associated PACAP(1-38)-induced vasodilation was now similar to that of SSM-associated VIP. This response was associated with a corresponding increase in alpha-helix content of both peptides in the presence of phospholipids. Collectively, these data indicate that for a larger amphipathic peptide, such as PACAP(1-38), greater kinetic energy or longer incubation period is required to optimize peptide-SSM interactions and amplify peptide bioactivity in vivo.     

Diethylene Glycol Distearate Embedding and Ultramicrotome Sectioning for Light Microscopy

Diethylene glycol distearate can be used as an embedding medium for light microscopy. Two infiltration changes of about 6 hr each in the melted wax (melting point 47-52 C) are required before the final embedding which is done in 00 gelatin capsules for sectioning in the ultramicrotome by the procedure used in electron microscopy. Serial sections 1-2 μ thick can be cut without difficulty. No cooling devices are necessary for trimming and sectioning at laboratory temperature. Sections rarely become detached from the slides. The staining characteristics of the tissues are the same as when embedded in paraffin. For fluorescence microscopy, essentially the same procedure is followed. Tissues are not distorted and the intracellular structures are well preserved.     

Formation of a unique crystal morphology for the poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymer

The crystallization behaviors of the poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymer with the PEG weight fraction of 0.50 (PEG(50)-PCL(50)) was studied by DSC, WAXD, SAXS, and FTIR. A superposed melting point at 58.5 degrees C and a superposed crystallization temperature at 35.4 degrees C were obtained from the DSC profiles running at 10 degrees C/min, whereas the temperature-dependent FTIR measurements during cooling from the melt at 0.2 degrees C/min showed that the PCL crystals formed starting at 48 degrees C while the PEG crystals started at 45 degrees C. The PEG and PCL blocks of the copolymer crystallized separately and formed alternating lamella regions according to the WAXD and SAXS results. The crystal growth of the diblock copolymer was observed by polarized optical microscope (POM). An interesting morphology of the concentric spherulites developed through a unique crystallization behavior. The concentric spherulites were analyzed by in situ microbeam FTIR, and it was determined that the morphologies of the inner and outer portions were mainly determined by the PCL and PEG spherulites, respectively. However, the compositions of the inner and outer portions were equal in the analysis by microbeam FTIR.     

Polyethylene Glycol Embedment for Histological Studies of Bean Seed Testa of Low Moisture Content

Light microscopic examination of the structure of seed testa of snap and semihard bean seeds with 6% and 12% moisture contents in paraffin sections was unsuccessful because of poor paraffin infiltration and subsequent separation of subjacent and palisade cell layers. We devised an alternative method using polyethylene glycol (PEG) as the embedding material. Specimens were killed and fixed in the usual manner. They were then run up through a graded series (25, 50, 75, 100%) of PEG 1000 to PEG 1450, and finally embedded in a mixture of PEG 1450 and 4000 (19:1 by weight). Transverse and longitudinal sections retained excellent morphological detail and were suitable for histological study. Sections temporarily stained with 0.025% thionin allowed good quality photomicrographs.     

Polyethylene glycol embedment for histological studies of bean seed testa of low moisture content

Light microscopic examination of the structure of seed testa of snap and semihard bean seeds with 6% and 12% moisture contents in paraffin sections was unsuccessful because of poor paraffin infiltration and subsequent separation of subjacent and palisade cell layers. We devised an alternative method using polyethylene glycol (PEG) as the embedding material. Specimens were killed and fixed in the usual manner. They were then run up through a graded series (25, 50, 75, 100%) of PEG 1000 to PEG 1450, and finally embedded in a mixture of PEG 1450 and 4000 (19:1 by weight). Transverse and longitudinal sections retained excellent morphological detail and were suitable for histological study. Sections temporarily stained with 0.025% thionin allowed good quality photomicrographs.     

Effect of pyrolysis temperature on composition, surface properties and thermal degradation rates of Brazil Nut shells

Changes in chemical and surface characteristics of Brazil Nut shells (Bertholletia excelsa) due to pyrolysis at different temperatures (350 degrees C, 600 degrees C, 850 degrees C) were examined. For this purpose, proximate and ultimate analyses, physical adsorption measurements of N2 (-196 degrees C) and CO, (25 degrees C) as well as samples visualisation by scanning electronic microscopy (SEM) were performed. Appreciable differences in the residue characteristics, depending markedly on the pyrolysis temperature, were observed. Release of volatile matter led to the development of pores of different sizes. Progressive increases in micropore development with increasing pyrolysis temperature took place, whereas a maximum development of larger pores occurred at 600 degrees C. Furthermore, kinetics measurements of Brazil Nut shells pyrolysis from ambient temperature up to 900 degrees C were performed by non-isothermal thermogravimetric analysis. A model taking into account the significant changes in the residue during pyrolysis, through an increase in the activation energy with temperature and solid conversion, were found to properly fit the kinetics data over the wide range of degradation investigated.     

The application of polyethylene glycol (PEG) to electron microscopy

The cytoplasm of cells from a variety of tissues has been viewed in sections (0.25-1 micrometers) devoid of any embedding resin. Glutaraldehyde- and osmium tetroxide-fixed tissues were infiltrated and embedded in a water-miscible wax, polyethylene glycol (PEG), and subsequently sectioned on dry glass or diamond knives. The PEG matrix was removed and the sections were placed on Formvarcarbon-polylysine- coated grids, dehydrated, dried by the critical-point method, and observed in either the high- or low-voltage electron microscope. Stereoscopic views of cells devoid of embedding resin present an image of cell utrastructure unobscured by electron-scattering resins similar to the image of whole, unembedded critical-point-dried or freeze-dried cultured cells observed by transmission electron microscopy. All organelles, including the cytoskeletal structures, are identified and appear not to have been damaged during processing, although membrane components appear somewhat less distinct. The absence of an embedding matrix eliminates the need for additional staining to increase contrast, unlike the situation with specimens embedded in standard electron-scattering resins. The PEG technique thus appears to be a valuable adjunct to conventional methods for ultrastructural analysis.     

Development of a simple embedding procedure allowing immunocytochemical localization at the ultrastructural level

Immunobed solution A is a water-soluble acrylic compound recently developed for immunocytochemical localization at the light microscopic level. In this study, we combined it with methyl methacrylate (MMA) to achieve sufficient hardness to obtain ultra-thin sections. Samples of platelets were dehydrated and embedded in the water-soluble acrylic mixture (WSAM). The embedding process was carried out at 4 degrees C and final polymerization was induced with either chemical (benzoyl peroxide) or physical (UV light) catalysts. Tubulin was localized at the ultrastructural level in sections embedded according to these two methods. Results were compared with those obtained in platelets processed in Lowicryl. Dehydration and embedding with the WSAM yielded a preservation of antigenicity similar to that obtained in Lowicryl. The new procedure benefits from the low temperature achieved during polymerization, providing good ultrastructural morphology and immunolocalization of protein antigens with the simplicity of a routine embedding procedure for light microscopy.     

Study of the synthesis, crystallization, and morphology of poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymers

Poly(ethylene glycol)-poly(epsilon-caprolactone) diblock copolymers PEG-PCL were synthesized by ring-opening polymerization of epsilon-caprolactone using monomethoxy poly(ethylene glycol) as the macroinitiator and calcium ammoniate as the catalyst. Obvious mutual influence between PEG and PCL crystallization was studied by altering the relative block length. Fixing the length of the PEG block (Mn = 5000) and increasing the length of the PCL block, the crystallization temperature of the PCL block rose gradually from 1 to about 35 degrees C while that of the PEG block dropped from 36 to -6.6 degrees C. Meanwhile, the melting temperature of the PCL block went up from 30 to 60 degrees C, while that of the PEG block declined from 60 to 41 degrees C. If the PCL block was longer than the PEG block, the former would crystallize first when cooling from a molten state and led to obviously imperfect crystallization of PEG and vice versa. And they both crystallized at the same temperature, if their weight fractions were equal. We found that the PEG block could still crystallize at -6.6 degrees C even when its weight fraction is only 14%. A unique morphology of concentric spherulites was observed for PEG5000-PCL5000. According to their morphology and real-time growth rates, it is concluded that the central and outer sections in the concentric spherulites were PCL and PEG, respectively, and during the formation of the concentric spherulite, the PEG crystallized quickly from the free space of the PCL crystal at the earlier stage, followed by outgrowing from the PCL spherulites in the direction of right angles to the circle boundaries until the entire area was occupied.     

Effect of high temperature and water stress on in vitro germination and growth in isolates of the entomopathogenic fungus Beauveria bassiana (Bals.) Vuillemin

In non-irrigated agricultural fields in tropical zones, high temperature and water stress prevail during the main cropping season. Natural epizootics of Beauveria bassiana on lepidopteran pests occur during winter. Application of B. bassiana during hot months when pest populations are at their climax may prove an effective management strategy. Therefore, 29 isolates of B. bassiana were tested for their ability to germinate and grow in temperature and water availability conditions prevailing during the pest season in these fields. The effect of temperature cycles with 8 h duration of high temperature fluctuating with 16 h duration of lower temperature (similar to field conditions); low water availability; and a combination of these two stress conditions was studied. Germination and growth assays were done at fluctuating temperature cycles of 32, 35, 38, and 42+/-1 degrees C (8 h)/25+/-1 degrees C (16 h) and in media with water stress created by 10, 20, 30, and 40% polyethylene glycol (PEG 6000). Assays set at a continuous temperature of 25+/-1 degrees C with no PEG in the medium served as controls. Stress was assessed as percentage germination or as growth relative to control. Isolates showing 90% growth relative to the control at temperature cycles including high temperatures of 35 and 38+/-1 degrees C were identified. One isolate (ARSEF 2860) had a thermal threshold above 43 degrees C. At 25 degrees C, all but one isolate of B. bassiana showed >90% growth relative to the control in 10% PEG (-0.45 MPa). Some isolates were found with >90% growth relative to control in medium having 30% PEG with water availability (1.33 MPa), nearly equivalent to that in soils which induce permanent wilting point of plants. When isolates that showed >90% growth relative to the control at both stress conditions, were stressed simultaneously, a decrease in growth was observed. Growth was reduced by approximately 20% at 35+/-1 degrees C (8 h)/25+/-1 degrees C (16 h) and 10% PEG and was affected to a greater degree in combinations of harsher stress conditions. The isolate ARSEF 2860 with a thermal threshold of >43 degrees C showed approximately 80% relative growth at a combined stress of 38+/-1 degrees C (8 h)/25+/-1 degrees C (16 h) and 10% PEG. These findings will aid the selection of isolates for use in field trials in hot or dry agricultural climates.     

Improved method for making high-affinity sections of soft tissue embedded in polyethylene glycol (PEG): its use in screening monoclonal antibodies

This article describes improvements in the immunohistologic technique for embedding highly hydrated embryonic tissue in polyethylene glycol 1000 (PEG)--a water-soluble wax of melting point 39 degrees C--and compares the PEG sections with frozen and polyester-wax sections. The main improvement ensures that relatively large PEG sections (8 X 3 mm) stretch out and adhere well to slides: a coat of albumen and glycerine is dried onto the slides and a fresh coat applied just before use. The embedding, sectioning, and mounting procedures, which are considerably faster than those for wax processing, have been developed for screening monoclonal antibodies against the differentiated neural crest cells in the anterior eyes of 9-day-old chick embryos. PEG sections of such eyes were a little fragile, but showed good cellular detail, similar to or better than in wax sections and considerably better than in frozen sections. The responses of PEG sections to the antibodies were far stronger than those of wax and marginally better than those of frozen sections. In one experiment using 125I-labeled rabbit anti-mouse antibody on sections previously treated with antibodies or antisera, PEG sections bound about five times as much label as wax sections and approximately 30% more than frozen sections. The main limitation of the technique is that, because of the softness of PEG, it only works well for embedding a limited range of tissues. Such PEG sections may, however, be useful for in situ hybridization as well as for immunohistochemistry.     

Cell fusion between temperature-sensitive mutants of a Drosophila melanogaster cell line.

Temperature-sensitive (ts) mutants were isolated in a cell line of Drosophila melanogaster, GM1, by ethyl methanesulfate treatment. Two of them, ts15 and ts58, formed colonies at 23 degrees C but not at 30 degrees when inoculated at densities of/or less than 10(5) cells per 60 X 15-mm dish. By using these ts mutants, cell fusion was attempted with polyethylene glycol (PEG) 6000. Several colonies per dish developed at 30 degrees C when different ts mutants were mixed, treated with PEG, and inoculated at a density of 10(4) cells per dish. Cells in some of the colonies thus developed were propagated and their temperature-sensitive character and karyotypes were studied. The results indicated that cell fusion could be induced with PEG and that the cells which formed colonies at 30 degrees C after PEG treatment were the hybrids in which the temperature-sensitive lesions in the mutants were complemented.     

Oil-encapsulating PEO-PPO-PEO/PEG shell cross-linked nanocapsules for target-specific delivery of paclitaxel

PEO-PPO-PEO/PEG shell cross-linked nanocapsules encapsulating an oil phase in their nanoreservoir structure was developed as a target-specific carrier for a water-insoluble drug, paclitaxel. Oil-encapsulating PEO-PPO-PEO/PEG composite nanocapsules were synthesized by dissolving an oil (Lipiodol) and an amine-reactive PEO-PPO-PEO derivative in dichloromethane and subsequently dispersing in an aqueous solution containing amine-functionalized six-arm-branched poly(ethylene glycol) by ultrasonication. The resultant shell cross-linked nanocapsules had a unique core/shell architecture with an average size of 110.7 +/- 9.9 nm at 37 degrees C, as determined by dynamic light scattering and transmission electron microscopy. Paclitaxel could be effectively solubilized in the inner Lipiodol phase surrounded by a cross-linked PEO-PPO-PEO/PEG shell layer. The paclitaxel-loaded nanocapsules were further conjugated with folic acid to achieve folate receptor targeted delivery. Confocal microscopy and flow cytometric analysis revealed that folate-mediated targeting significantly enhanced the cellular uptake and apoptotic effect against folate receptor overexpressing cancer cells. The present study suggested that these novel nanomaterials encapsulating an oil reservoir could be potentially applied for cancer cell targeted delivery of various water-insoluble therapeutic and diagnostic agents.     

Comparative studies of Feulgen hydrolysis for DNA. I. Influence of different fixatives and polyethylene glycols

We compared the Feulgen hydrolysis curves (37 degrees C, 5 M HCl) of human blood lymphocytes fixed by the following four methods: 96% ethanol, 60 min at 20 degrees C; ethanol-acetone, 1:1, 120 min at 4 degrees C; ethanol-glacial acetic acid mixture (3:1), containing 2% of formaldehyde (EAF), 90 min at 20 degrees C; and ethanol-glacial acetic acid (3:1), 60 min followed by 5% chrome alum solution for 360 min at 20 degrees C. The best results were obtained with EAF-fixation, with respect to the highest amount of DNA-Schiff complex at the peak point of the curve, the longest "plateau" region and the smallest scattering of DNA-Schiff amount values along the "plateau". With other types of fixation the addition of polyethylene glycol (PEG) 6,000 to the hydrolysis solution resulted in modification of the shape of the hydrolysis curve so that it became nearly similar to the EAF-curve. The effect of PEG6000 on the EAF-curve was minimal. Slides fixed by ethanol were used for a comparison of polyethylene glycols with m.w. 1,500, 6,000 and 20,000. The longest "plateau" was obtained with PEG6000. The only effect of PEG1500 was a dramatic increase of DNA-Schiff amount at the peak point. PEG20000 had no significant effect on the shape of the hydrolysis curve. The results are discussed in terms of Kjellstrand's "chain with a stable structure" model of Feulgen hydrolysis.     

A new method for transfer of polyethylene glycol-embedded tissue sections to silanated slides for immunocytochemistry

Polyethylene glycol (PEG) is an excellent embedding medium for immunohistochemical studies. It provides structural preservation superior to frozen sections and increased sensitivity of antigen detection compared with paraffin sections. One limitation of PEG embedment is that PEG sections are difficult to handle and adhere poorly to glass slides. Here we present a simple and effective method for embedding tissues in PEG and transferring the resultant sections onto silanated glass slides. In addition, a method for silver enhanced colloidal gold immunostaining was combined with common dye staining to demonstrate the excellent structure preservation and sensitive antigen detection. Bovine chorionic membrane was fixed with Bouin's fixative, embedded in polyethylene glycol (PEG) 1500, cut into 5-microns sections, flattened over agarose blocks (10 x 10 x 2 mm3), and blotted onto Digene silanated slides. Slides were then washed in PBS, which removed the PEG and agarose blocks. Tissue sections were immunocytochemically stained with dilute antiserum raised in a rabbit against purified bovine placental retinol binding protein (bpRBP). Sections were washed and incubated with 1-nm colloidal gold-labeled goat anti-rabbit IgG. The immunogold particles were enhanced by silver staining (IGSS). Specimens were observed and photographed with an Olympus epipolarization microscope. The new method offered excellent morphological preservation of cell structure and the epipolarization microscopy provided high sensitivity for detection of specific immunogold-silver particles.     

Thermal stability studies of a globular protein in aqueous poly(ethylene glycol) by (1)H NMR

The reversible folding destabilization of hen lysozyme has been confirmed by a melting temperature (T(m)) decrease in aqueous poly(ethylene glycol) (PEG). The percent denatured, extracted from the histidine 15 C2H (H15 C2H) native and denatured peak areas from 500-MHz one-dimensional proton nuclear magnetic resonance (1D (1)H NMR) spectra in D(2)O, was analyzed through denaturation temperatures at 0% and 20% (w/w) PEG 1000. The lysozyme (3.5 mM) T(m) decreased by 4.2 degrees C and 7.1 degrees C in 20% (w/w) PEG 1000 at pH 3.8 and 3.0, respectively. The T(m) decreased with increasing lysozyme concentration. Additionally, the temperature-induced resonance migrations of 17 protons from 8 residues indicate that the native lysozyme structure undergoes temperature-induced conformational changes. The changes were essentially identical in both 0% and 20% (w/w) PEG 1000 at both pH 3.0 and 3.8. This small, local restructuring of the hydrophobic box region may be a manifestation of temperature-dependent solution hydrophobicity, whereas active-site cleft fluctuations may be due to the inherent active-site flexibility. The lysozyme structure in PEG at 35 degrees C was determined to be essentially native from the (1)H nuclear Overhauser effect spectroscopy (NOESY) fingerprint regions. Additionally, lysozyme chemical shifts, from 1D spectra, in PEG 200, 300, and 1000 at 35 degrees C and various concentrations were essentially identical, further confirming that the conformation remains native in various PEG solutions. (c) 1996 John Wiley & Sons, Inc.