Three dimensional polyvinyl alcohol scaffolds modified with collagen for HepG2 cell culture.

The creation of in vitro functional hepatic tissue simulating micro environmental niche of the native liver is a keen area of research due to its demand in bioartificial liver. However, it is still unclear how to maintain benign cell function while achieving the sufficient cell quantity. In this work, we aim to prepare a novel scaffold for the culture of HepG2 cells, a liver cell line, by modifying polyvinyl alcohol (PVA) scaffold with collagen (COL). PVA is a kind of synthetic biostable polymer with high hydrophilicity in the human body, has been widely used in the biomedical field. However, the use of PVA is limited in cell cultures due to lack of biologically active functional groups. In this study, amino silane (KH-550), glutaraldehyde and native type I collagen were used to modify three-dimensional PVA scaffold to establish a suitable composite scaffold for hepatocyte culture. Three types of composite scaffolds were prepared for different collagen content, named as PVA/COL (0.2%), PVA/COL (0.5%) and PVA/COL (0.8%), respectively. The composite scaffolds were characterized by SEM, XPS, FTIR, MS, porosity estimation and water contact angle measurement. The PVA/COL (0.8%) scaffolds had the highest collagen content of 12.13%. The composite scaffold showed high porosity with interconnected pores. Furthermore, the biocompatibility between HepG2 cells and scaffolds was evaluated by the ability of cell proliferation, albumin secretion, as well as urea synthesis. The coating of collagen on PVA scaffolds promoted hydrophilicity and HepG2 cell adhesion. Additionally, enhanced cell proliferation, increased albumin secretion and urea synthesis were observed in HepG2 cells growing on collagen-coated three-dimensional PVA scaffolds.

Optimization of aqueous enzymatic method for Camellia sinensis oil extraction and reuse of enzymes in the process.

Aqueous enzymatic extraction of Camellia sinensis oil was studied. The results suggested that saponin removal pretreatment assisted by ultrasound was effective in decreasing emulsification and in enhancing the free oil recovery. After 70% isopropanol extraction for 30 min under ultrasound, the residue of C. sinensis seeds was further hydrolyzed with free cellulase and Alcalase for 5 h, and calcium ions were concurrently added during enzymatic hydrolysis (nCa2+: nsaponin = 1:2), and free oil recovery up to 94.14% was obtained. Separate immobilization and co-immobilization of Alcalase and cellulase were performed by alginate entrapment combined with glutaraldehyde crosslinking. Specific activity and recovery of activity for Alcalase and cellulase were acceptable. After immobilization, Alcalase and cellulase exhibited higher activity at a wider pH and temperature range. Reuse experiments of immobilized enzymes were conducted. The deactivation kinetics immobilized enzymes were simulated and half-life of immobilized enzyme was estimated. The results indicated that a magnetic supporter facilitated the recovery of immobilized enzymes from tea seed slurry, and that immobilized Alcalase and cellulase had good reusability.

Quantification of protein mobility and associated reshuffling of cytoplasm during chemical fixation.

To understand cellular functionalities, it is essential to unravel spatio-temporal patterns of molecular distributions and interactions within living cells. The technological progress in fluorescence microscopy now allows in principle to measure these patterns with sufficient spatial resolution. However, high resolution imaging comes with long acquisition times and high phototoxicity. Therefore, physiological live cell imaging is often unfeasible and chemical fixation is employed. Yet, fixation methods have not been rigorously investigated, in terms of pattern preservation, at the resolution at which cells can now be imaged. A key parameter for this is the time required until fixation is complete. During this time, cells are under unphysiological conditions and patterns decay. We demonstrate here that formaldehyde fixation takes more than one hour for cytosolic proteins in cultured cells. Other small aldehydes, glyoxal and acrolein, did not perform better. Associated with this, we found a distinct displacement of proteins and lipids, including their loss from cells. Fixations using glutaraldehyde were faster than four minutes and retained most cytoplasmic proteins. Surprisingly, autofluorescence produced by glutaraldehyde was almost completely absent with supplementary addition of formaldehyde without compromising fixation speed. These findings indicate, which cellular processes can actually be reliably imaged after a certain chemical fixation.

Determination of total creatine kinase activity in blood serum using an amperometric biosensor based on glucose oxidase and hexokinase.

Creatine kinase (CK: adenosine-5-triphosphate-creatine phosphotransferase) is an important enzyme of muscle cells; the presence of a large amount of the enzyme in blood serum is a biomarker of muscular injuries, such as acute myocardial infarction. This work describes a bi-enzyme (glucose oxidase and hexokinase based) biosensor for rapid and convenient determination of CK activity by measuring the rate of ATP production by this enzyme. Simultaneously the biosensor determines glucose concentration in the sample. Platinum disk electrodes were used as amperometric transducers. Glucose oxidase and hexokinase were co-immobilized via cross-linking with BSA by glutaraldehyde and served as a biorecognition element of the biosensor. The biosensor work at different concentrations of CK substrates (ADP and creatine phosphate) was investigated; optimal concentration of ADP was 1mM, and creatine phosphate - 10 mM. The reproducibility of the biosensor responses to glucose, ATP and CK during a day was tested (relative standard deviation of 15 responses to glucose was 2%, to ATP - 6%, to CK - 7-18% depending on concentration of the CK). Total time of CK analysis was 10 min. The measurements of creatine kinase in blood serum samples were carried out (at 20-fold sample dilution). Twentyfold dilution of serum samples was chosen as optimal for CK determination. The biosensor could distinguish healthy and ill people and evaluate the level of CK increase. Thus, the biosensor can be used as a test-system for CK analysis in blood serum or serve as a component of multibiosensors for determination of important blood substances. Determination of activity of other kinases by the developed biosensor is also possible for research purposes.

The effects of the covalent attachment of 3-(4-hydroxy-3,5-di-tert-butylphenyl) propyl amine to glutaraldehyde pretreated bovine pericardium on structural degeneration, oxidative modification, and calcification of rat subdermal implants.

Bioprosthetic heart valves (BHV) fabricated from glutaraldehyde pretreated heterograft materials, porcine aortic valves or bovine pericardium (BP), are widely used in cardiac surgery. BHV progressively fail in clinical use due to structural degeneration. Previously we reported that dityrosine, an oxidized amino acid, was present in failed clinical BP-BHV explants; unimplanted BP had no detectable dityrosine. In the same studies BP were demonstrated in vitro to be susceptible to oxidative damage, that could be mitigated with BP covalently modified with the antioxidant, 3-(4-hydroxy-3,5-di-tert-butylphenyl)propyl amine (DBP). The present studies compared in rat subdermal implants glutaraldehyde pretreated BP to BP modified with either DBP or the chemical reactions used to link DBP. All BP explants regardless of DBP demonstrated reduced hydroxyproline and increased digestibility by collagenase. However, the DBP-BP explants showed significant inhibition of reduced explant shrink temperatures (an index of crosslinking) as compared with control BP. Significant mitigation of calcification was observed in both the BP-DBP and chemically modified explants as compared with BP. Dityrosine was not detectable in the 90 day explants. It is concluded that rat subdermal BP implants undergo both calcific and noncalcific structural degeneration, but without the formation of dityrosine, unlike clinical BP explants.

Impact of glutaraldehyde on in vivo colon-specific release of resveratrol from biodegradable pectin-based formulation.

Despite potential therapeutic efficacy of resveratrol on colitis and colorectal cancer, rapid absorption and metabolism at the upper gastro-intestinal (GI) tract prevent its clinical application. To overcome this, we attempted to develop colon-specific multi-particulate calcium-pectinate (Ca-pectinate) formulations of resveratrol. However, they were unable to prevent premature drug release at the upper GI tract. Thus, glutaraldehyde (Glu) was used for further cross-linking of the pectin chains. The formulation conditions and procedure were optimized from the in vitro drug release study. The optimized formulation was subjected to in vivo pharmacokinetic study in rats and compared with the unmodified Ca-pectinate and suspension formulation of resveratrol. Spherical particles (∼1 mm diameter) with high drug encapsulation were produced. Low cross-linking solution pH (1.5), minimum Glu concentration (2.5%) and cross-linking time (2 h) were crucial to exhibit colon-specific drug release. As Glu was added in the cross-linking solution, cross-linking between pectin chains and Glu occurred simultaneously during Ca-pectinate network formation, which appeared as a cost-effective formulation technique. Most importantly, the pharmacokinetic study demonstrated in vivo colon-specific drug release from the optimized formulation, while faster drug release was observed from the unmodified and suspension formulations. Hence, the developed formulation has potential to be used as colon-specific delivery system of resveratrol.

Recombinant CPE fused to tumor necrosis factor targets human ovarian cancer cells expressing the claudin-3 and claudin-4 receptors.

Using gene expression profiling, others and we have recently found that claudin-3 (CLDN3) and claudin-4 (CLDN4) are two of the most highly and consistently up-regulated genes in ovarian carcinomas. Because these tight junction proteins are the naturally occurring receptors for Clostridium perfringens enterotoxin (CPE), in this study, we used the COOH-terminal 30 amino acids of the CPE (CPE(290-319)), a fragment that is known to retain full binding affinity but have no cytolytic effect, to target tumor necrosis factor (TNF) to ovarian cancers. We constructed a pET32-based vector that expressed the fusion protein, designated here as CPE(290-319)-TNF, in which CPE(290-319) was fused to TNF at its NH(2)-terminal end. Western blotting confirmed presence of both CPE(290-319) and TNF in the fusion protein. The TNF component in CPE(290-319)-TNF was 5-fold less potent than free TNF as determined by a standard L-929 TNF bioassay. However, the CPE(290-319)-TNF was >6.7-fold more cytotoxic than free TNF to 2008 human ovarian cancer cells, which express both CLDN3 and CLDN4 receptors. shRNAi-mediated knockdown of either CLDN3 or CLDN4 expression in 2008 markedly attenuated the cytotoxic effects of CPE(290-319)-TNF. The fusion construct was efficiently delivered into target cells and located in both cytosol and vesicular compartments as assessed by immunofluorescent staining. We conclude that CPE(290-319) effectively targeted TNF to ovarian cancer cells and is an attractive targeting moiety for development of CPE-based toxins for therapy of ovarian carcinomas that overexpress CLDN3 and CLDN4.

A simple and direct pre-embedding technique for ultrastructure of scarce biological specimens.

A simple and rapid technique for pre-embedding scarce biological specimens for Transmission electron microscopy (TEM) is reported. It is based on pre-embedding biological samples in bovine serum albumin (BSA) and bis-acrylamide (BA), cross-linked and polymerized with paraformaldehyde, glutaraldehyde, ammonium persulfate and Temed. Pre-embedding in BSA and BA offers several advantages over traditional pre-embedding techniques for TEM including the ability to visualize the sample and a more resistant matrix. This results in more reproducible and consistent analysis. It can be applied to tissues, cells, and subcellular structures handled as pellets or suspensions. In addition, use of the pre-embedding matrix for light microscopy is reported. The ability to pre-embed scarce biological specimens efficiently and reproducibly provides a valuable way to study and characterize cytological tissues such as biopsies or cystic and amniotic fluid cells.

A biosensor based on Bay leaf (Laurus nobilis L.) tissue homogenate: improvement of the stability characteristics by a simple bio-imprinted technique.

Although enzymes are effective biocatalysts that are widely used in biosensors, a major drawback that hampers many of these biotechnological applications of enzymes is their limited stability. Applications that use very pure, high value proteins need to employ effective stabilization technology, primarily due to cost considerations and availability of the proteins used. For this purpose, interest in bio-imprinting techniques increases because it allows stability characteristics of enzymes to be improved. In this study, a bio-imprinted Bay leaf (Laurus nobilis L.) tissue homogenate biosensor was devised by a very simple way. For this purpose, the enzymes, polyphenol oxidases in the bay leaf tissue, were first complexed by using their competitive inhibitor, thiourea, in aqueous medium and then this enzyme was immobilized on gelatin by crosslinking with glutaraldehyde on a Clark-type oxygen electrode surface. Similarly, noncomplexed polyphenol oxidase with thiourea was also immobilized on a Clark-type oxygen electrode in the same conditions. The aim of the study was to prepare a new biosensor-based Bay leaf tissue homogenate and to improve the stability characteristics such as thermal stability, pH stability, and storage stability, of the biosensor by bio-imprinting method. The results showed that this simple technique should be effectively used to improve the stabilities of a biosensor.

Sulfhydryl-based tumor antigen-carrier protein conjugates stimulate superior antitumor immunity against B cell lymphomas.

Therapeutic vaccination of B cell lymphoma patients with tumor-specific Ig (idiotype, or Id) chemically coupled to the immunogenic foreign carrier protein keyhole limpet hemocyanin (KLH) using glutaraldehyde has shown promising results in early clinical trials, and phase III trials are underway. However, glutaraldehyde Id-KLH vaccines fail to elicit anti-Id immune and clinical responses in many patients, possibly because glutaraldehyde reacts with lysine, cysteine, tyrosine, and histidine residues, damaging critical immunogenic epitopes. A sulfhydryl-based tumor Ag-carrier protein conjugation system using maleimide chemistry was used to enhance the efficacy of Id-KLH vaccines. Maleimide Id-KLH conjugates eradicated A20 lymphoma from most tumor-bearing mice, whereas glutaraldehyde Id-KLH had little efficacy. Maleimide Id-KLH elicited tumor-specific IgG Abs and T cells, with CD8(+) T cells being the major effectors of antilymphoma immunity. Maleimide Id-KLH vaccines also demonstrated superior efficacy in 38C13 and BCL-1 lymphoma models, where Abs were shown to be critical for protection. Importantly, standard glutaraldehyde Id-KLH conjugation procedures could result in "overconjugation" of the tumor Ag, leading to decreased efficacy, whereas the heterobifunctional maleimide-based conjugation yielded potent vaccine product regardless of conjugation duration. Under lysosomal processing conditions, the Id-carrier protein linkage was cleavable only after maleimide conjugation. Maleimide KLH conjugation was easily performed with human Igs analogous to those used in Id-KLH clinical trials. These data support the evaluation of sulfhydryl-based Id-KLH vaccines in lymphoma clinical trials and possibly the use of tumor Ag-carrier protein vaccines for other cancers.

Inhibition of prostate cancer metastasis by administration of a tissue vaccine.

Immunotherapy by vaccination represents a novel method for treatment of cancer. In this regard, vaccines with the broadest possible menu of relevant antigens stand the greatest chance of success. Tissue vaccines are composed of material harvested directly from tumors and contain not only antigens associated with neoplastic epithelium, but also those that may be unique to in vivo growth and antigens associated with the tumor stroma. To test the hypothesis that a tissue vaccine, produced by glutaraldehyde fixation of harvested syngeneic prostate tumors (GFT vaccine), could be used for treatment of prostate cancer, male Lobund-Wistar (LW) rats were treated with methylnitrosourea (MNU) and testosterone propionate to induce autochthonous prostate tumors. Tumor-bearing rats were randomly assigned to one of three treatment groups: no treatment (11 rats); vaccination with media (10 rats); or vaccination with the GFT vaccine (19 rats). Vaccination was given initially with Freund's complete adjuvant and booster doses were given with incomplete Freund's adjuvant every week until the time of euthanasia. There were no significant differences in mean tumor weight between groups; however, GFT-vaccinated rats had a prolonged survival time; and 4/19 (21%) GFT-vaccinated rats were found to be tumor-free compared to none of the untreated or media-treated controls. Further, pulmonary metastasis occurred in only 5/15 (33%) of GFT-vaccinated rats compared to 10/11 (91%) and 10/10 (100%) of untreated and media-vaccinated controls, respectively. Supernatants of cultured splenocytes from similarly media- and GFT-vaccinated rats demonstrated significant (P < 0.001) increases in IFN-gamma and TNF-alpha from splenocytes of GFT-vaccinated rats, suggesting that GFT vaccination stimulates a Th1 response. In summary, treatment of tumor-bearing rats with a tissue vaccine stimulated a protective immune response that resulted in complete tumor regression in 21% of animals and reduced the number of animals with any evidence of metastasis by nearly 70%. These results suggest that tissue vaccines may be useful for the treatment of prostate cancer.

An easy method using glutaraldehyde-introduced fluorescence for the microscopic analysis of plant biotrophic interactions.

The method introduced in this article makes use of the glutaraldehyde-induced auto-fluorescence of proteins after cross-linking with glutaraldehyde for the analysis of cellular and sub-cellular structures. Because the interface of biotrophic interactions is rich in proteins, the method presented is particularly suitable for the analysis of such interactions; we have exemplified its usefulness by analyzing (1) the root feeding sites induced in roots from Arabidopsis thaliana by the root-knot nematode Meloidogyne incognita; (2) leaves from Cucurbita pepo infected by powdery mildew and (3) roots from Nicotiana tabacum colonized by the arbuscular mycorrhizal fungus Glomus intraradices. The use of confocal and multi-photon laser scanning microscopy allows three-dimensional reconstructions from optical sections of complex biotrophic interactions. In the case of root-knot nematode feeding sites, our method enabled us to simultaneously study the development of the plant xylem elements (using lignin auto-fluorescence), the nematode feeding site and the nematode itself.

[Analysis of properties of collagen membranes before and after crosslinked].

OBJECTIVE: To compare the properties of collagen membranes before and after crosslinked and to establish the foundation of application of collagen membranes. METHODS: Fresh bovine tendons were separated and collagen was extracted by washing, smashing and acetic acid dissolving. The collagen protein was determined by ultraviolet spectrophotometer and its characteristics were analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), wavelength scanning and amino acids detecting. Collagen membranes were produced by lyophilization. And then the biocharacteristics of the membranes before and after glutaraldehyde crosslinked were compared. BMSCs separated from volunteer's bone marrow were seeded on collagen membranes before and after crosslinked by 2 x 10(3) in 100 microL medium, seven days after culture, the absorption spectrum of BMSCs was examined, and BMSCs were observed by scanning electron microscope (SEM). RESULTS: The contents of collagen protein were 2 mg/mL. The maximum absorption wave length appeared at about 230 nm. SDS-PAGE suggested that molecular weight of main bands was more than 66.2 x 10(3), the same as collagen marker from calf skin. There were 21.47% glycine, 12.04% praline and 10.18% hydroxyproline. No tryptophan was found. Before crosslinked, collagen membranes were in shape of white sponges and with big holes and the range of pH value was from 4.5 to 5.0. SEM showed reticular conformation and pore structure of collagen membranes, but the bore diameter was bigger. Their water-absorbing capacity was 61 times as much as their weight. The mechanical strength was 210 g/cm3. The dissolution time of collagenase was 90 minutes. After crossl inked, collagen membranes became thin, colorless, semi-transparent and compact with better tenacity. Under SEM, compact collagen fiber appeared reticular. There was lower water-absorbing capacity and pH value ranged from 6.5 to 7.0. The mechanical strength was 3,400 g/cm3 and the dissolution time of collagenase became longer. BMSCs could grow better either on before-crosslinked collagen membranes or on after-crosslinked ones. CONCLUSION: As biomaterial scaffolds, after crosslinked collagen membranes were better than before-crosslinked ones.

Yessotoxin induces the accumulation of altered E-cadherin dimers that are not part of adhesive structures in intact cells.

We have studied the alteration induced by yessotoxin in the E-cadherin-catenin system of epithelial cells by stabilizing the protein-protein interactions in oligomers, through the introduction of covalent bonds between subunits in vitro and in vivo. The E-cadherin-catenin complexes that we have stabilized by crosslinking comprise multiple forms of dimeric, trimeric, tetrameric and hexameric complexes, with different subunit compositions. A 1-day treatment of MCF-7 cells with yessotoxin resulted in an increase in cellular levels of the complexes including a 100kDa fragment of E-cadherin (ECRA100), with a relative increase in cellular E-cadherin .ECRA100 heterodimers, as opposed to the E-cadherin homodimer that represents the core structure of the E-cadherin-catenin system of adhesive structures in normal cells. The high MW oligomers of cell adhesive structures, in turn, were not appreciably altered by cell treatment with yessotoxin. Most of these oligomers partitioned in a fraction that cannot be solubilized by non-ionic detergents after crosslinking of intact cells. Yessotoxin treatment did not significantly alter the levels of ECRA100 in the Triton X-100 resistant fraction of plasma membrane, but increased the relative abundance of ECRA100 in the Triton X-100 soluble pool of crosslinked cells. We have concluded that cell exposure to yessotoxin leads to increased cellular contents of E-cadherin .ECRA100 heterodimers that are not participating to cell adhesive structures but are located in other membranous fractions of intact cells.

The interaction of DNR and glutaraldehyde with cell membrane proteins leads to morphological changes in erythrocytes.

In this study, the effects of DNR and glutaraldehyde on isolated erythrocyte membrane proteins were examined. For this purpose, SDS-gel electrophoresis was carried out. Additionally, analyses of the disturbances in erythrocyte shape and size, accompanied by the application of flow cytometry and microscopy examination, were undertaken. The amount of DNR linked to erythrocyte cell membranes was measured by a fluorimetric technique. It was observed that glutaraldehyde caused in concentration dependent manner an increase of percent of DNR linked to cell membrane proteins. After this incorporation, perturbations in the protein content of cell membranes were observed. The protein aggregates and changes in the level of spectrin, actin and band 3 protein were noted. Due to the changes in spectrin, which is mainly responsible for maintenance of the discocyte shape of erythrocytes, flow cytometry and microscopy techniques were used to control the size and shape of erythrocytes after treatment with DNR and glutaraldehyde. The disturbances in the shape and size of erythrocytes were observed for all tested concentrations of glutaraldehyde. For all tested concentrations of glutaraldehyde, the changes were statistically significant.

Effects of glutaraldehyde exposure on human health.

Glutaraldehyde (GA) is widely used in the industrial, scientific and biomedical fields. Many adverse health effects on humans have been reported in association with biomedical uses of GA, with 2-3.5% aqueous GA solution generally used for cold sterilization and GA exposure ranges of 0.001 to 2.6 ppm for this type of use. GA is metabolized extensively to CO(2), but urinary excretion of it is low. Sensory irritant effects, sensitization of skin and respiratory organs and other symptoms have been reported among endoscopy nurses and medical radiation technologists. The prevalence of chronic bronchitis and nasal symptoms in humans is significantly correlated with peak concentrations of GA exposure. The extent of primary skin irritation depends on the duration and site of contact, and the severity of symptoms is dose-related. Chronic inhalation affects the nose and respiratory tract, and lesions become severe with prolonged duration of exposure. Increases in neither mortality nor tumor incidence have been found in workers with less than 0.2 ppm GA exposure, no evidence of carcinogenic activity has been obtained in experimental animal studies. There has been no clear evidence of genetic toxicity of GA in either in vitro or in vivo studies, and neither developmental nor reproductive toxicity has been found in humans or animals. To prevent hazards from GA exposure, use of closed-system, fully automated washing machines is recommended, since numerous symptoms have been found in individuals with less than 0.05 ppm GA exposure, the recommended peak exposure limit in many countries.

Regulation of water permeability in rabbit conjunctival epithelium by anisotonic conditions.

Effects of unilateral exposure to anisotonic conditions on diffusional water permeability of the isolated rabbit conjunctiva were determined. A segment of the bulbar-palpebral conjunctiva was mounted between Ussing-type hemichambers under short-circuit conditions. Unidirectional water fluxes (J(dw)) were measured in either direction by adding (3)H(2)O to one hemichamber and sampling from the other. Electrical parameters were measured simultaneously. J(dw) were determined under control isosmotic conditions and after introduction of either hyper- or hypotonic solutions against the tear or stromal sides of the preparations. In each of these four separate conditions, the anisotonic medium produced an approximately 20-30% reduction in J(dw) across the tissue, with the exception that to obtain such reduction with increased tonicity from the stromal side (medium osmolality increased by adding sucrose), conditions presumptively inhibiting regulatory volume increase mechanisms (e.g., pretreatment with amiloride and bumetanide) were also required. All reductions in J(dw) elicited by the various anisotonic conditions were reversible on restoration of control tonicity. In experiments in which preparations were pretreated with the protein cross-linking agent glutaraldehyde, anisotonicity-elicited reductions in J(dw) were not observed. Such reductions were also not observed in the presence of HgCl(2), implying the involvement of aquaporins. However, it is possible that the mercurial may be toxic to the epithelium, preventing the tonicity response. Nevertheless, from concomitant changes in transepithelial electrical resistance, as well as [(14)C]mannitol fluxes, [(14)C]butanol fluxes, and Arrhenius plots, arguments are presented that the above effects are best explained as a cell-regulated reduction in membrane water permeability that occurs at the level of water-transporting channels. Presumably both apical and basolateral membranes can downregulate their water permeabilities as part of a protective mechanism to help maintain cell volume.

Insights into the structure of human cytomegalovirus large terminase subunit pUL56.

Terminases are a class of proteins which catalyze the generation of unit-length genomes during DNA packaging. These essential proteins are conserved throughout the herpesviruses and many double-stranded DNA bacteriophages. We have determined the structure of the large terminase subunit pUL56 of human cytomegalovirus, a highly pathogenic virus, to 2.6 nm resolution. Image analysis of purified pUL56 suggests that the molecule exists as a dimer formed by the association of two ring-like structures positioned on top of each other and connected by a pronounced density on one side. The 3D reconstruction of pUL56 provides first structural insights into the active protein.

Iron-regulatory proteins DmdR1 and DmdR2 of Streptomyces coelicolor form two different DNA-protein complexes with iron boxes.

In high G+C Gram-positive bacteria, the control of expression of genes involved in iron metabolism is exerted by a DmdR [divalent (bivalent) metal-dependent regulatory protein] in the presence of Fe2+ or other bivalent ions. The dmdR1 and dmdR2 genes of Streptomyces coelicolor were overexpressed in Escherichia coli and the DmdR1 and DmdR2 proteins were purified to homogeneity. Electrophoretic mobility-shift assays showed that both DmdR1 and DmdR2 bind to the 19-nt tox and desA iron boxes forming two different complexes in each case. Increasing the concentrations of DmdR1 or DmdR2 protein shifted these complexes from their low-molecular-mass form to the high-molecular-mass complexes. Formation of the DNA-protein complexes was prevented by the bivalent metal chelating agent 2,2'-dipyridyl and by antibodies specific against the DmdR proteins. Cross-linking with glutaraldehyde of pure DmdR1 or DmdR2 proteins showed that DmdR1 forms dimers, whereas DmdR2 is capable of forming dimers and probably tetramers. Ten different iron boxes were found in a search for iron boxes in the genome of S. coelicolor. Most of them correspond to putative genes involved in siderophore biosynthesis. Since the nucleotide sequence of these ten boxes is identical (or slightly different) with the synthetic DNA fragment containing the desA box used in the present study, it is proposed that DmdR1 and DmdR2 bind to the iron boxes upstream of at least ten different genes in S. coelicolor.

Preliminary results with the use of an albumin-glutaraldehyde tissue adhesive in lung surgery.

BACKGROUND: The purpose of this study was to test the performance of an albumin-glutaraldehyde tissue adhesive, BioGlue(r) Surgical Adhesive (BioGlue) in the sealing of air leaks from pulmonary parenchyma and bronchopleural fistulas. MATERIAL/METHODS: Between March 2000 and November 2001 BioGlue was applied in 38 randomly selected patients, who underwent 39 operations. The mean age was 51.4 years (range 19 to 75 years). A median of 5 cc of BioGlue was used per patient (range 5 to 20 cc). The operations included 36 thoracotomies, 2 video-assisted thoracoscopies and one rigid bronchoscopy. RESULTS: The duration of air leak ranged from 0 to 2 days with a median of 1 day. The duration of total (air and fluid) chest tube drainage ranged from 1 to 12 days with a median of 3 days. Complications were observed in 3 patients (8%) and included atelectasis in one and residual space in 2. Three patients died because of preexisting respiratory failure unrelated to BioGlue application. Hospitalization ranged from 4 to 16 days with a median of 6 days and was prolonged in some patients because of their primary disease (empyema, bronchopleural fistula, etc.). CONCLUSIONS: The use of BioGlue proved to be safe and effective in the sealing of lung lacerations and in preventing air leakage from suture or staple lines in emphysematous lungs. It was also successful in sealing bronchopleural fistulas when applied either intra-bronchially through the rigid bronchoscope or during thoracotomy.