A gel-sandwich technique for the qualitative and quantitative determination of dehydrogenases in the enzyme histochemistry

Summary A gel-sandwich technique for the histochemical demonstration of dehydrogenases is introduced with LDH set up as an example. Especially suitable, of the gels examined, for this technique is 1.5% W/V agar-agar low gel strength. In it several reaction ingredients for the histochemical reaction are dissolved. Considering LDH the following gel composition showed good results: 1.5% W/V agar-agar low gel strength, 5 mM TNBT in 150 l DMF, 120 mM L-lactate, 3–5 mM NAD⁺, 10 mM amytal, 22,4–32×10^–5 M Meldola Blue, 160 mM soldium phosphate buffer pH 7.6 (total solution of 1 ml). After the solidification of the gel, gel-bars were frozen with CO₂-snow. The 40–80 m thick gel slices were gained in the cryostat. Of the three different arrangement possibilities of the gel slices and the tissue-sections a sandwich arrangement (cover-gel slice — tissue section — ground-gel slice) produced the best results. The enzyme reaction is started by thawing of the gel slices (together with the tissue sections) and by putting them between the hotplate and the evaporator-head-piece, especially developed for this technique. The gel slices also remain in combination with the tissue sections after the reaction.The influence of the gel in combination with the electron carrier Meldola Blue on the spontaneous reduction rates of ditetrazolium salts in day light, were examined as well as the diffusion rates of TNBT and NADH out of gel slices and the influence of DMF and DMSO on the LDH activity.This technique prevents both, the loss of enzymes and the loss of reduction equivalents. There are given presuppositions for qualitative and quantitative histochemical investigations as well. The advantages of the new gel technique are discussed.     

Adipose tissue can be generated in vitro by using adipocytes from human fat tissue mesenchymal stem cells seeded and cultured on fibrin gel sheet

The current study has developed an innovative procedure to generate ex novo fat tissue by culturing adipocytes from human fat tissue mesenchymal stem cells (hFTMSCs) on fibrin gel sheet towards applications in medicine and cosmetology. Fibrin gel has been obtained by combining two components fibrinogen and thrombin collected by human peripheral blood. By this procedure it was possible to generate blocks of fibrin gel containing adipocytes within the gel that show similar features and consistency to human fat tissue mass. Results were assessed by histological staining methods, fluorescent immune-histochemistry staining as well photos by scanning electron microscopy (SEM) to demonstrate the adhesion and growth of cells in the fibrin gel. This result opens a real possibility for future clinical applications in the treatment of reconstructive and regenerative medicine where the use of stem cell may eventually be a unique solution or in the field of aesthetic medicine where autograft fat stem cells may grant for a safer and better outcome with long lasting results.     

A Phase 1 Randomized,Open Label,Rectal Safety,Acceptability, Pharmacokinetic,and Pharmacodynamic Study of Three Formulations of Tenofovir 1% Gel (the CHARM-01 Study)

Objectives

The CHARM-01 study characterized the safety, acceptability, pharmacokinetics (PK), and pharmacodynamics (PD) of three tenofovir (TFV) gels for rectal application. The vaginal formulation (VF) gel was previously used in the CAPRISA 004 and VOICE vaginal microbicide Phase 2B trials and the RMP-02/MTN-006 Phase 1 rectal safety study. The reduced glycerin VF (RGVF) gel was used in the MTN-007 Phase 1 rectal microbicide trial and is currently being evaluated in the MTN-017 Phase 2 rectal microbicide trial. A third rectal specific formulation (RF) gel was also evaluated in the CHARM-01 study.

Methods

Participants received 4 mL of the three TFV gels in a blinded, crossover design: seven daily doses of RGVF, seven daily doses of RF, and six daily doses of placebo followed by one dose of VF, in a randomized sequence. Safety, acceptability, compartmental PK, and explant PD were monitored throughout the trial.

Results

All three gels were found to be safe and acceptable. RF and RGVF PK were not significantly different. Median mucosal mononuclear cell (MMC) TFV-DP trended toward higher values for RF compared to RGVF (1136 and 320 fmol/10⁶ cells respectively). Use of each gel in vivo was associated with significant inhibition of ex vivo colorectal tissue HIV infection. There was also a significant negative correlation between the tissue levels of TFV, tissue TFV-DP, MMC TFV-DP, rectal fluid TFV, and explant HIV-1 infection.

Conclusions

All three formulations were found to be safe and acceptable. However, the safety profile of the VF gel was only based on exposure to one dose whereas participants received seven doses of the RGVF and RF gels. There was a trend towards higher tissue MMC levels of TFV-DP associated with use of the RF gel. Use of all gels was associated with significant inhibition of ex vivo tissue HIV infection.

Trial Registration

ClinicalTrials.gov NCT01575405     

Decision and management algorithms to address patient and food and drug administration concerns regarding breast augmentation and implants

During the U.S. Food and Drug Administration's advisory panel hearings to evaluate the premarket approval for conventional silicone gel implants on October 14 and 15, 2003, panel members and patient advocate representatives focused on four specific areas of concern: reoperation rates in primary breast augmentation; levels, depth, and methods of patient education and informed consent; modes, frequency, and management of silicone gel implant device failures, including management of "silent" ruptures; and methods of monitoring and managing symptoms or symptom complexes that may or may not be associated with connective tissue disease or other undefined symptom complexes. These concerns, with a reported 20 percent reoperation rate for primary augmentation within just 3 years, and a lack of concise, definitive management protocols addressing these areas of concern may have contributed to the Food and Drug Administration's rejection of the premarket approval, despite the panel's recommendation for approval. This article presents decision and management algorithms that have been used successfully for 7 years in a busy breast augmentation practice (Tebbetts and Tebbetts). The algorithms have been further expanded and refined by a group of surgeons with diverse experiences and expertise to address the following clinical situations that coincide with concerns expressed by patients and the Food and Drug Administration: implant size exchange, grade III to IV capsular contracture, infection, stretch deformities (implant bottoming or displacement), silent rupture of gel implants, and undefined symptom complexes (connective tissue disease or other). In one practice (Tebbetts and Tebbetts) that uses the TEPID system (tissue characteristics of the envelope, parenchyma, and implant and the dimensions and fill distribution dynamics of the implant), implant selection is based on quantified patient tissue characteristics, pocket selection is based on quantified soft-tissue coverage, and anatomic saline implants have fill volumes that are designed to minimize shell collapse and fold fatigue; in this practice, the algorithms contributed to a 3 percent overall reoperation rate in 1662 reported cases with up to 7 years of follow-up, compared with a 20 percent reoperation rate at 3 years in the 2003 premarket approval study.     

Fabrication of Biologically Derived Injectable Materials for Myocardial Tissue Engineering

This protocol provides methods for the preparation of an injectable extracellular matrix (ECM) gel for myocardial tissue engineering applications. Briefly, decellularized tissue is lyophilized, milled, enzymatically digested, and then brought to physiological pH. The lyophilization removes all water content from the tissue, resulting in dry ECM that can be ground into a fine powder with a small mill. After milling, the ECM powder is digested with pepsin to form an injectable matrix. After adjustment to pH 7.4, the liquid matrix material can be injected into the myocardium. Results of previous characterization assays have shown that matrix gels produced from decellularized pericardial and myocardial tissue retain native ECM components, including diverse proteins, peptides and glycosaminoglycans. Given the use of this material for tissue engineering, in vivo characterization is especially useful; here, a method for performing an intramural injection into the left ventricular (LV) free wall is presented as a means of analyzing the host response to the matrix gel in a small animal model. Access to the chest cavity is gained through the diaphragm and the injection is made slightly above the apex in the LV free wall. The biologically derived scaffold can be visualized by biotin-labeling before injection and then staining tissue sections with a horse radish peroxidase-conjugated neutravidin and visualizing via diaminobenzidine (DAB) staining. Analysis of the injection region can also be done with histological and immunohistochemical staining. In this way, the previously examined pericardial and myocardial matrix gels were shown to form fibrous, porous networks and promote vessel formation within the injection region.Download video file.^{(70M, mov)}     

A Multi-Compartment Single and Multiple Dose Pharmacokinetic Comparison of Rectally Applied Tenofovir 1% Gel and Oral Tenofovir Disoproxil Fumarate

This Phase 1, randomized, two-site (United States), double-blind, placebo-controlled study enrolled 18 sexually abstinent men and women. All received a single 300-mg dose of oral tenofovir disoproxil fumarate (TDF) and were then randomized 2∶1 to receive single and then seven daily rectal exposures of vaginally-formulated tenofovir (TFV) 1% gel or a hydroxyethyl cellulose (HEC) placebo gel. Blood, colonic biopsies and rectal and vaginal mucosal fluids were collected after the single oral TDF, the single topical TFV gel dose, and after 7 days of topical TFV gel dosing for extracellular analysis of TFV and intracellular analysis of the active metabolite tenofovir diphosphate (TFVdp) in peripheral blood mononuclear cells (PBMCs) and isolated mucosal mononuclear cells (MMC), including CD4+ and CD4- cell subsets. With a single rectal dose, TFV plasma concentrations were 24–33 fold lower and half-life was 5 h shorter compared to a single oral dose (p = 0.02). TFVdp concentrations were also undetectable in PBMCs with rectal dosing. Rectal tissue exposure to both TFV and TFVdp was 2 to 4-log₁₀ higher after a single rectal dose compared to a single oral dose, and after 7 daily doses, TFVdp accumulated 4.5 fold in tissue. TFVdp in rectal tissue homogenate was predictive (residual standard error, RSE  = 0.47) of tissue MMC intracellular TFVdp concentration, with the CD4+ cells having a 2-fold higher TFVdp concentration than CD4- cells. TFV concentrations from rectal sponges was a modest surrogate indicator for both rectal tissue TFV and TFVdp (RSE  = 0.67, 0.66, respectively) and plasma TFV (RSE  = 0.38). TFV penetrates into the vaginal cavity after oral and rectal dosing, with rectal dosing leading to higher vaginal TFV concentrations (p<0.01).

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00984971","term_id":"NCT00984971"}}NCT00984971     

Quantification of human neutrophil motility in three-dimensional collagen gels. Effect of collagen concentration.

Leukocytes must migrate through tissues to fulfill their role in the immune response, but direct methods for observing and quantifying cell motility have mostly been limited to migration on two-dimensional surfaces. We have now developed methods for examining neutrophil movement in a three-dimensional gel containing 0.1 to 0.7 mg/ml rat tail tendon collagen. Neutrophil-populated collagen gels were formed within flat glass capillary tubes, permitting direct observation with light microscopy. By following the tracks of individual cells over a 13.5-min observation period and comparing them to a stochastic model of cell movement, we quantified cell speed within a given gel by estimating a random motility coefficient (mu) and persistence time (P). The random motility coefficient changed significantly with collagen concentration in the gel, varying from 1.6 to 13.3 x 10(-9) cm2/s, with the maximum occurring at a collagen gel concentration of 0.3 mg/ml. The methods described may be useful for studying tissue dynamics and for evaluating the mechanism of cell movement in three-dimensional gels of extracellular matrix (ECM) molecules.     

Pituitary protein lipolytic factor(s): Partial purification by isoelectric focusing (IEF)

Tests were made on highly purified bovine peptidal pituitary factors for lipolytic activity on rat adipose tissue. The lipolytic protein factor from bovine pituitary glands was isolated and characterized by ethanol precipitation, gel filtration, and isoelectric focusing. The supernatant of the homogenized tissue was precipitated with ethanol, the precipitate was lyophilized and resuspended in HCl 1 mmole/liter, the insoluble was discarded, and the supernatant lyophilized. A solution of the lyophilized fraction was fractionated by gel filtration on Sephadex G-75. The bands with lipolytic activity were pooled and lyophilized. Lipolytic activity was determined by incubation of epididimal rat adipose tissue and by successive measurements of free fatty acids and glycerol released in the incubation medium. The gel filtration with the highest lipolytic activity (20.4 µequiv/g per 4 hr and 17.0 µmole/g per 4 hr glycerol) was submitted to isoelectric focusing. The gel filtration still appeared highly heterogeneous, but most of the lipolytic activity was concentrated in the protein band at pH 8.6.     

Immunological identification of uncoupling protein in interscapular "brown" adipose tissue of suckling and adult pipistrelle bats (Pipistrellus pipistrellus).

1. Interscapular adipose tissue of suckling and adult pipistrelle bats was examined for the presence of the 32,000 Mr "uncoupling protein" diagnostic of brown adipose tissue. 2. Following separation by SDS-polyacrylamide gel electrophoresis, mitochondrial proteins were blotted onto nitrocellulose and probed for uncoupling protein with an anti-(ground squirrel uncoupling protein) serum. 3. Immunoreactivity consistent with the presence of uncoupling protein was found in all samples of adipose tissue mitochondria from both suckling and adult bats. 4. It is concluded that interscapular adipose tissue in pipistrelle bats exhibits the critical biochemical criterion for being designated functionally "brown".     

简单快速的DNA银染和胶保存方法

本文介绍了一套简单快速的DNA银染以及胶保存的方法,整个过程仅需10~15分钟,而且背景浅,条带清楚,灵敏度高,稳定性好。胶保存采用双层玻璃纸夹心法,可长久地保存胶显色时的原貌。以常规PAG胶检测和HLA的SSCP分型为例,利用该套方法进行了银染以及胶的保存,均得到了满意的结果。该方法具有推广价值。 Abstract:This paper introduced the simple and rapid methods of silver staining and gel preservation.It was taken only about 10 and 15 minutes to stain a gel.The background of gel was light,the bands were clear,the sensibility was high and the stabilization was well by the method of silver staining.The gel preservation adopted a method named two-layer transparent plastic paper "Sandwich" which could keep the gel with primitive colors for a long time.The methods were used on PAG checking and SSCP typing of HLA and the results were satisfactory.The set of methods are expected to be widely used in laboratories.     

The 65-kDa cytosolic protein associated with warm temperature acclimation in goldfish,Carassius auratus

Goldfish Carassius auratus were acclimated to either 10 or 30°C for a minimum of 5 weeks. A 65-kDa protein specific to warm-temperature-acclimated fish was extracted from the gel with 70% formic acid after two-dimensional electrophoresis of the muscle cytoplasmic protein fraction. The 65-kDa protein thus prepared to homogeneity was used to raise specific antibodies in rabbit by conventional methods. The antibody produced exhibited specific reaction with a protein having the same molecular weight from brain and liver tissue, suggesting that the 65-kDa protein is a ubiquitous cytosolic component in warm-acclimated goldfish. When water temperature was increased from 20 to 30°C over a 20-h period, a prominent amount of the 65-kDa protein was observed in muscle tissue extracts within 5 days of additional rearing; this was demonstrated by immunoblotting with the specific antibody. The N-terminal amino acid sequence of the 65-kDa protein was determined as Asp-Glu-Pro-Gln-Gly-His-Gln-His (or Asp)-Glu-Leu, differing from that of a family of known heat-shock proteins having about 70 kDa in molecular mass (hsp 70). No interaction between ATP and the 65-kDa protein revealed by ATP-agarose affinity chromatography further confirmed the different properties of the 65-kDa protein from those of hsp 70.Abbreviations ATP adenosine 5-triphosphate - hsp heat-shock protein(s) - IgG immunoglobulin G - mRNA messenger ribonucleic acid - PMSF phenylmethylsulphonyl fluoride - PVDF polyvinylidene difluoride - SDS sodium dodecyl sulphate - SDS-PAGE SDS-polyacrylamide gel electrophoresis     

Mapping an initiation region of DNA replication at a single-copy chromosomal locus in Drosophila melanogaster cells by two-dimensional gel methods and PCR-mediated nascent-strand analysis: multiple replication origins in a broad zone.

We have mapped an initiation region of DNA replication at a single-copy chromosomal locus in exponentially proliferating Drosophila tissue culture cells, using two-dimensional (2D) gel replicon mapping methods and PCR-mediated analysis of nascent strands. The initiation region was first localized downstream of the DNA polymerase alpha gene by determining direction of replication forks with the neutral/alkaline 2D gel method. Distribution of replication origins in the initiation region was further analyzed by using two types of 2D gel methods (neutral/neutral and neutral/alkaline) and PCR-mediated nascent-strand analysis. Results obtained by three independent methods were essentially consistent with each other and indicated that multiple replication origins are distributed in a broad zone of approximately 10 kb. The nucleotide sequence of an approximately 20-kb region that encompasses the initiation region was determined and searched for sequence elements potentially related to function of replication origins.     

P15-T SameSpots: Validating a Novel Approach to 2D Electrophoresis Image Analysis

2D-PAGE experiments can provide a powerful means of investigating protein expression behavior in a cell or tissue across different disease states or other experimental conditions. Traditional analysis of 2D experiments typically requires a large amount of post-detection editing in order to prepare the data for statistical research. With the application of SameSpots, all protein spots are fully matched and thus no missing values exist. This enables a more robust statistical exploration of the data with a reduction in subjective editing.The SameSpots workflow has a semi-automatic gel alignment step and uses the arcsinh transform introduced by Huber et al.¹ for inter-gel calibration and variance stabilization (VSN). To validate these techniques we repeated the work of Nishihara and Champion² and Karp and Lilley.³ The results of this study demonstrate that gel alignment has no adverse affect on spot volume quantitation, and we also show that VSN outperforms traditional normalization and variance stabilizing methods used in 2D gel analysis.     

Biochemical properties,homology, and genetic variation of Drosophila “nonspecific” esterases

The biochemical properties and tissue distribution of two major, soluble "nonspecific" esterases have been studied in Drosophila melanogaster, D. pseudoobscura, and related species. The "alpha-like" activity is due to a monomer enzyme (MW congruent to 60 kd) having a nonspecific tissue distribution, which was inhibited by p-hydroxymercuribenzoate (5 X 10(-4)M) plus eserine (1 X 10(-5)M) and was relatively unstable during polyacrylamide gel electrophoresis. Electrophoretograms of this enzyme could be enhanced by treating gels with beta-mercaptoethanol before staining. This procedure allowed the identification of a new alpha-esterase (Est-4) in D. pseudoobscura. The "beta-like" esterase activity (EC 3.1.1.1) is due to a dimer (MW congruent to 120 kd) in most Drosophila species. D. melanogaster and its siblings (D. simulans and D. mauritiana) were exceptions in which this enzyme had an unusual tissue distribution (increased activity in the male reproductive system) and was a monomer (MW congruent to 60 kd). Differences in the genetic variability of these esterases are discussed and interpreted by a population expansion model rather than by differences in biochemical properties of enzyme forms.     

Experimental study of tissue response to ruptured gel-filled mammary prostheses.

Miniature "soft gel" filled breast prostheses were implanted into rats, and half of these were purposefully ruptured upon insertion. At sacrifice of the animals later, the position of the exuded gel, the variability of the capsule thickness, and the extent of the capsule formation were evaluated. The capsule thickness was significantly increased around the ruptured prostheses, in response to the exuded gel. Also, the gel migrated through the fibrous capsule and into the surrounding tissue, eliciting an inflammatory response there to produce capsule thickening.     

Correlation between Compartmental Tenofovir Concentrations and an Ex Vivo Rectal Biopsy Model of Tissue Infectibility in the RMP-02/MTN-006 Phase 1 Study

Objectives

This study was designed to assess the dose-response relationship between tissue, blood, vaginal and rectal compartment concentrations of tenofovir (TFV) and tenofovir diphosphate (TFVdp) and ex vivo rectal HIV suppression following oral tenofovir disoproxil fumarate (TDF) and rectal administration of TFV 1% vaginally-formulated gel.

Design

Phase 1, randomized, two-site (US), double-blind, placebo-controlled study of sexually-abstinent males and females.

Methods

Eighteen participants received a single 300 mg exposure of oral TDF and were then randomized 2∶1 to receive a single then seven-daily rectal exposures of TFV 1% gel (40 mg TFV per 4 ml gel application) or hydroxyethyl-cellulose (HEC) placebo gel. Blood and rectal biopsies were collected for pharmacokinetic TDF and TFVdp analyses and ex vivo HIV-1 challenge.

Results

There was a significant fit for the TFVdp dose-response model for rectal tissue (p = 0.0004), CD4⁺ _MMC (p<0.0001), CD4⁻ _MMC (p<0.0001), and Total_MMC (p<0.0001) compartments with r² ranging 0.36–0.64. Higher concentrations of TFVdp corresponded with lower p24, consistent with drug-mediated virus suppression. The single oral treatment failed to provide adequate compartment drug exposure to reach the EC₅₀ of rectal tissue TFVdp predicted to be necessary to suppress HIV in rectal tissue. The EC₅₀ for CD4⁺ _MMC was within the single topical treatment range, providing evidence that a 1% topical, vaginally-formulated TFV gel provided in-vivo doses predicted to provide for 50% efficacy in the ex vivo assay. The 7-daily topical TFV gel treatment provided TFVdp concentrations that reached EC₉₀ biopsy efficacy for CD4⁻ _MMC, CD4⁺ _MMC and Total_MMC compartments.

Conclusion

The TFVdp MMC compartment (CD4+, CD4− and Total) provided the best surrogate for biopsy infectibility and the 7-daily topical TFV gel treatment provided the strongest PK profile for HIV suppression.ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00984971","term_id":"NCT00984971"}}NCT00984971.     

Thiophilic-interaction chromatography of enzymatically active tissue prostate-specific antigen (T-PSA) and its modulation by zinc ions

Prostate-specific antigen (PSA) is a serine protease secreted both by normal prostate glandular epithelial cells and prostate cancer cells. We explored "thiophilic-interaction chromatography" (TIC) to isolate tissue prostate-specific antigen (T-PSA) from fresh human prostate cancer tissue harvested by radical prostatectomy for the purpose to characterize T-PSA for its enzymatic activity and sensitivity to zinc ions. We have shown, for the first time, that T-PSA has strong affinity for the thiophilic gel (T-gel). The average recovery of T-PSA from T-gel is over 87%. The presence of PSA in the column eluate was confirmed by ELISA and SDS/PAGE. Western blot developed with monoclonal antibody to PSA revealed that T-PSA was predominantly in the "free" form having a molecular weight of 33 kDa. Furthermore, T-PSA was found to be enzymatically active. T-PSA was found to be less enzymatically active as compared to seminal plasma PSA. The inhibition of enzymatic activity of both f-PSA and T-PSA over a wide range of concentrations of Zn(2+) ions (10nM to 50 microM) was comparable. In contrast, the enzymatic activity of chymotrypsin, another serine-protease, was affected differently. At higher concentrations of Zn(2+) (10 microM and higher) the enzymatic activity of chymotrypsin was inhibited, whereas, at lower concentrations of Zn(2+) (5 microM and lower), the enzymatic activity was enhanced.     

Is the "mammalian" brown fat-specific mitochondrial uncoupling protein present in adipose tissues of birds?

1. Mitochondria were isolated from the furcular, subcutaneous, abdominal, nape and lateral adipose tissue depots of five species of bird (pheasant, Japanese quail, pigeon, house sparrow and great tit) acclimatized to the Northern winter. 2. Mitochondrial proteins were separated by SDS-polyacrylamide gel electrophoresis, blotted onto nitrocellulose membranes, and probed for the presence of the 32,000-33,000 Mr uncoupling protein characteristic of "mammalian" brown adipose tissue, using an anti-(ground squirrel uncoupling protein)serum. 3. Immunoreactivity consistent with uncoupling protein was not detected in mitochondria from any of the avian adipose tissues. Immunoreactivity was, however, evident in mitochondria from perirenal or interscapular adipose tissue from a range of mammals--rats, mice, golden hamsters, Orkney voles, wood mice, pipistrelle bats, wood lemmings, and newborn lambs, cattle, reindeer and red deer. 4. These results provide biochemical evidence that "mammalian-like" thermogenic brown adipose tissue is absent from avian species; adipose tissues in birds appear to be functionally "white".     

A procedure for DNA and RNA transfer to membrane filters avoiding weight-induced gel flattening.

We describe a technique of rapid (within 1-2 h) transfer of DNA and RNA from agarose gels to nitrocellulose or nylon membrane filters. It is characterized by nearly complete elimination of mechanical action on the gel (a thin layer of liquid is placed over the gel and, filtering through the gel into a stack of paper towels beneath, it transfers nucleic acids onto the filter under the gel). This "descending" transfer, as opposed to the widely used "ascending" Southern transfer, reduces the transfer time (to about 1 h) with equal or higher quality of the hybridization signal. The comparison of transfer kinetics by the both methods shows that (a) the Southern transfer of large size DNA fragments proceeds quicker than it has been thought so far and is almost complete within 4 h; (b) the descending transfer has an advantage over the ascending one in the rate of transfer (1-2 h) and its efficiency; and (c) the time of transfer may become a critical parameter upon using a filter with an apparently low retention capacity (Hybond N, Amersham) that is manifested by a decreased signal at longer than optimal transfer times.     

Caprolactonic poloxamer analog: PEG-PCL-PEG

The aqueous solution of poly(ethylene glycol)-poly(caprolactone)-poly(ethylene glycol) (PEG-PCL-PEG) triblock copolymers (> 15. wt. %) undergoing "clear sol-gel-turbid sol" transition as the temperature increases from 20 to 60 degrees C has been developed. Light scattering and 13C NMR study suggested that the transition mechanisms are the micellar aggregation for the clear sol to gel transition (lower transition), whereas the increase in PCL molecular motion for gel to turbid sol transition (upper transition). In contrast to the previous thermogelling biodegradable polymers with a sticky paste morphology, the powder form of the PEG-PCL-PEG triblock copolymers makes it easy to handle and allows fast dissolution in water. Therefore, the lyophilization into a powder form followed by facile reconstitution was possible. This system is believed to be promising for drug delivery, cell therapy, and tissue engineering.