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PubMed2003

Antisense treatment against Ki-67 mRNA inhibits proliferation and tumor growth in vitro and in vivo.

The Ki-67 protein is tightly regulated and depends on the proliferative status of a cell. It is present in the nuclei of proliferating cells but absent in resting cells. Since transformation of malignant cells is frequently associated with high cell proliferation and since proliferation is tightly associated with the Ki-67 protein labeling index, this antigen may represent a potential target for cancer therapy. In the present study we determined the ability of a phosphorothioate antisense oligodeoxyribonucleotide (ODN) targeted against Ki-67 mRNA to inhibit tumor cell proliferation specifically in cell culture, in multicellular 3-dimensional spheroids (MCS) and in subcutaneous murine tumor models. Antisense treatment of 1 myeloid and different epithelial tumor cell lines in suspension and monolayer culture, respectively, resulted in specific reduction of Ki-67 mRNA and protein, inhibition of proliferation and increased apoptotic cell death. Multicellular human bladder carcinoma spheroids lost their 3-dimensional structure and underwent cell death after incubation with antisense oligonucleotides. The growth of subcutaneous syngeneic prostatic (p = 0.05) and transitional cell tumors (p = 0.001) in immunocompetent mice was significantly inhibited in antisense-treated animals. From these findings we conclude that antisense inhibition of Ki-67 protein expression may be a rational approach in anticancer therapy.

Adenocarcinoma↗
PubMed1977

Platelet membrane defects in Glanzmann's thrombasthenia. Evidence for decreased amounts of two major glycoproteins.

Platelets from patients with Glanzmann's thrombasthenia have a distinct molecular alteration of the plasma membrane surface, namely decreased amounts of a major glycoprotein designated as IIb (apparent mol wt 142,000). To identify other possible surface defects of thrombasthenic platelets, we labeled the membrane polypeptides of normal and thrombasthenic platelets by two different techniques: lactoperoxidase-catalyzed iodination and galactose oxidase oxidation, followed by reduction with tritiated sodium borohydride. Labeling patterns were determined after the polypeptides were separated by two-dimensional polyacrylamide gel electrophoresis. Before the second dimension was run, platelet samples were incubated with a reducing agent, beta-mercapto-ethanol, to cleave the disulfide bonds of certain glycoproteins; the resulting changes in electrophoretic mobility permitted better resolution of individual molecules. Comparison of the labeled polypeptides of normal and thrombasthenic samples after reduction indicated decreased labeling of two major glycoproteins in thrombasthenic platelets: IIb and III (apparent mol wt 114,000). The relative proportions of radioactivity incorporated by these polypeptides were about 60 and 80% less than control values, respectively. With either Coomassie Blue or periodic acid-Schiff's reagent, glycoprotein III stained much less intensely in thrombasthenic compared to normal samples, indicating that the observed labeling deficit was caused by a decreased concentration of the molecule rather than steric inaccessibility on the membrane surface. Analysis of normal plasma membranes by affinity chromatography showed that glycoprotein IIb has receptors for lectin from Lens culinaris, the common lentil, whereas III does not. We conclude that a characteristic feature of Glanzmann's thrombasthenia is a decreased concentration of two discrete glycoproteins in the platelet plasma membrane.

Blood Platelet Disorders↗
PubMed2005

Identification of thioredoxin h-reducible disulphides in proteomes by differential labelling of cysteines: insight into recognition and regulation of proteins in barley seeds by thioredoxin h.

Using thiol-specific fluorescence labelling, over 30 putative target proteins of thioredoxin h with diverse structures and functions have been identified in seeds of barley and other plants. To gain insight at the structural level into the specificity of target protein reduction by thioredoxin h, thioredoxin h-reducible disulphide bonds in individual target proteins are identified using a novel strategy based on differential alkylation of cysteine thiol groups by iodoacetamide and 4-vinylpyridine. This method enables the accessible cysteine side chains in the thiol form (carbamidomethylated) to be distinguished from those inaccessible or disulphide bound form (pyridylethylated) according to the mass difference in the peptide mass maps obtained by matrix-assistend laser desorption/ionisation-time of flight mass spectrometry. Using this approach, in vitro reduction of disulphides in recombinant barley alpha-amylase/subtilisin inhibitor (BASI) by barley thioredoxin h isoform 1 was analysed. Furthermore, the method was coupled with two-dimensional electrophoresis for convenient thioredoxin h-reducible disulphide identification in barley seed extracts without the need for protein purification or production of recombinant proteins. Mass shifts of 15 peptides, induced by treatment with thioredoxin h and differential alkylation, identified specific reduction of nine disulphides in BASI, four alpha-amylase/trypsin inhibitors and a protein of unknown function. Two specific disulphides, located structurally close to the alpha-amylase binding surfaces of BASI and alpha-amylase inhibitor BMAI-1 were demonstrated to be reduced to a particularly high extent. For the first time, specificity of thioredoxin h for particular disulphide bonds is demonstrated, providing a basis to study structural aspects of the recognition mechanism and regulation of target proteins.

Allergens↗
PubMed1987

Inhibition of bacteriochlorophyll synthesis in Rhodobacter sphaeroides subsp. denitrificans grown in light under denitrifying conditions.

The inclusion of nitrate or nitrite in cultures of Rhodobacter spaeroides subsp. denitrificans grown heterotrophically in light depressed the formation of bacteriochlorophyll a. The pigment biosynthesis was inhibited at the stage of the reduction of chlorophyllide (chlorin) to bacteriochlorophyllide (tetrahydroporphyrin) since 3-hydroxyethylchlorophyllide a accumulated in the culture medium. The addition of exogenous 5-aminolevulinic acid to these cultures resulted in a complete restoration of bacteriochlorophyll synthesis accompanied by the accumulation of 3-vinylbacteriopheophorbide. This indicates that under these conditions bacteriochlorophyll was formed via an alternative route, in which the reduction of chlorins to tetrahydroporphyrins precedes modifications of the C-3 side chain. The multiple forms of 5-aminolevulinic acid synthase were purified from cells grown with and without nitrate. Antibodies against these proteins were raised in rabbits and used in enzyme-linked immunosorbent assays for various forms of 5-aminolevulinic acid synthase. In denitrifying cells, the amount and activity of fraction I of the enzyme was reduced by approximately 40 and 30%, respectively. Partly active enzymes from both types of cells were activated by cystine trisulfide.

5-Aminolevulinate Synthetase↗
PubMed2004

Presynaptic dopaminergic dysfunction in schizophrenia: a positron emission tomographic [18F]fluorodopa study.

CONTEXT: The dopamine overactivity hypothesis of schizophrenia remains one of the most influential theories of the pathophysiology of the illness. Radiotracer brain imaging studies are now directly testing aspects of the overactivity hypothesis. OBJECTIVE: To assess presynaptic dopaminergic function in a large cohort of patients with schizophrenia by means of [18F]fluorodopa uptake and a high-sensitivity 3-dimensional positron emission tomograph. We predicted elevations in striatal [18F]fluorodopa uptake and reductions in prefrontal cortical [18F]fluorodopa uptake in patients with schizophrenia. DESIGN: Case-control study. SETTING: Research institute investigation recruiting hospital outpatients. PATIENTS: Sixteen male medicated hospital outpatients with a DSM-IV diagnosis of schizophrenia (mean age, 38 years) and 12 age-matched male volunteers free of psychiatric and neurologic illness. INTERVENTION: [18F]fluorodopa positron emission tomographic scanning. MAIN OUTDOME MEASURE: [18F]fluorodopa uptake constant Ki measured with statistical parametric mapping and region-of-interest analyses. RESULTS: Statistical parametric mapping (P<.05 corrected) and region-of-interest analyses (P<.01) showed increased [18F]fluorodopa uptake, confined primarily to the ventral striatum in patients with schizophrenia. No reductions in prefrontal cortical [18F]fluorodopa uptake Ki were seen in the statistical parametric mapping and region-of-interest analyses, although dorsal anterior cingulate [18F]fluorodopa Ki correlated with performance on the Stroop Color-Word Test in both groups. CONCLUSIONS: As in studies in unmedicated patients, presynaptic striatal dopamine dysfunction is present in medicated schizophrenic patients, adding further in vivo support for dopamine overactivity in the illness.

Adult↗
PubMed2004

[Stereotactic irradiation of lung tumors].

Stereotactic irradiation of lung tumors is a relatively new technique aiming at increased applicable radiation doses by a reduction of normal tissue involvement. As a result of adequate patient immobilization, three dimensional treatment planning and highly precise target point definition, the safety margins generally used in conventional radiotherapy can be reduced significantly. Increased fraction doses, which have an amplified biological effect, can be used and lead to a shortening of the overall treatment time. Especially for localized early-stage non-small-cell lung cancer the achieved dose escalation is proven to correlate with an increased local tumor control. But also the utilization for the therapy of lung metastases of solid tumors is possible. In spite of small patient cohorts and limited long-term data compared to conventional radiotherapy the results are promising.

Dose Fractionation, Radiation↗
PubMed1995

Estimation of overall pulmonary function after irradiation using dose-effect relations for local functional injury.

PURPOSE: To predict the pulmonary function 3-4 months after irradiation for malignant lymphoma from the three-dimensional (3-D) dose distribution. METHODS: Dose-effect relations for the relative reduction of local perfusion (Q) and local ventilation (V), were calculated in 25 patients, using correlated SPECT (Single Photon Emission Computed Tomography) and CT data. By combining the 3-D dose distribution of an individual patient with the dose-effect relations averaged over all patients, the average reduction of local Q and V (i.e., the overall response parameters) in the whole lung was estimated for each patient. Correlation coefficients were calculated between these overall response parameters and the change in standard lung function tests. In addition, the relation between the overall response parameters and the incidence of radiation pneumonitis was determined. RESULTS: The overall response parameter for perfusion was correlated with the change in standard lung function tests, with correlation coefficients varying between 0.53 (p = 0.007) and 0.71 (p < 0.001) for the change of Vital Capacity and Forced Expiratory Volume at 1 s, respectively. For the overall response parameter for ventilation similar correlations were observed. Four out of the 25 patients developed radiation pneumonitis; in these four patients the overall response parameter for perfusion was on average somewhat higher (13.2 +/- 1.4% (1 standard error of the mean)) than in patients without radiation pneumonitis (10.5 +/- 1.0%), but this difference was not significant. A higher incidence of radiation pneumonitis was observed for larger values of the overall response parameter for perfusion; in patient groups with an overall response parameter for perfusion of 0-5%, 5-10%, 10-15%, and 15-20%, the incidence of radiation pneumonitis was 0 (0/1), 10 (1/10), 13 (1/8) and 33% (2/6), respectively. CONCLUSION: By combining the 3-D dose distribution with the average dose-effect relations for local perfusion or ventilation, an overall response parameter can be calculated prior to irradiation, which is predictive for the radiation-induced change in the overall pulmonary function, and possibly for the incidence of radiation pneumonitis, in this group of patients.

Adolescent↗
PubMed1989

Calculation of complication probability factors for non-uniform normal tissue irradiation: the effective volume method.

An estimation of normal tissue complication probability factors is important, particularly for evaluating 3-dimensional treatment plans. A method has been developed to calculate complication probability factors for non-uniformly irradiated normal organs using dose volume histograms and complication probabilities for uniform partial organ irradiation. In the effective volume method each volume element of the histogram is considered independently and subject to a power law dose volume relationship. Thus, a non-uniform dose volume histogram is reduced to a uniform one with an effective volume, and a dose equal to the maximum dose to the organ. The complication probability is then obtained from known complication probabilities for uniform partial organ irradiation. The effective volume histogram transformation method is shown to obey various boundary conditions, and is illustrated by comparing probability calculations for alternative 3-dimensional treatment plans for the pelvis. In addition, the limitations of this histogram reduction method are discussed and compared to other calculational techniques. The use of probability factor calculations in treatment plan evaluation, and their role in numerical scoring is explored.

Humans↗
PubMed2002

Combined hypermethylation and chromosome loss associated with inactivation of SSI-1/SOCS-1/JAB gene in human hepatocellular carcinomas.

We previously demonstrated using restriction landmark genomic scanning-based 2-dimensional genome electrophoresis method decreased results of 16 primary hepatocellular carcinomas (HCCs) revealed reduction of intensity of 60 NotI-landmark spots, and increase in five spots that were frequently observed in HCCs. Most frequently decreased spot (14/16 HCCs) was identified to it corresponds to a gene encoding SSI-1, a JAK-binding protein (SSI-1/SOCS-1/JAB) that regulated the JAK/STAT signal transduction pathway. This signaling pathway is important for relaying signals from various cytokines outside the cell to the inside. Expression level of SOCS-1 messenger RNA was markedly suppressed in 50% of HCCs (4/8). Loss of heterozygosity at the SSI-1 gene, was found in all cases with aberrant expression. Methylation analysis of the CpG-rich regions of SSI-1 gene revealed hypermethylation of these regions. In an additional series of methylation analysis using 30 HCCs, 16 (53%) showed hypermethylation of the gene. These results indicate that the SSI-1 gene is silenced in a substantial portion of HCC though the combined mechanisms of methylation of either 5' or exon CpG rich regions and by a chromosomal loss of the remaining allele.

Carcinoma, Hepatocellular↗
PubMed1993

Redox potentials of active-site bis(cysteinyl) fragments of thiol-protein oxidoreductases.

The active sites of thiol-protein oxidoreductases consist of the characteristic Cys-X-X-Cys motif, and the redox potentials of these enzymes reflect the propensity of the bis(cysteinyl) sequence portion for disulfide loop formation. Thereby, as is known from comparing the three-dimensional (3D) structures of thioredoxin and glutaredoxin in the reduced and oxidized state, reduction of the disulfide bond is accompanied by minimal perturbation of the backbone folding of the active sites. In order to estimate the sequence-dependent intrinsic free energy of formation of the active-site disulfide loops in oxidoreductases, synthetic fragments corresponding to the sequences 31-38, 10-17, 134-141, and 34-41 of thioredoxin, glutaredoxin, thioredoxin reductase, and protein disulfide isomerase (PDI), respectively, were analyzed for their tendency to form 14-membered rings. For this purpose thiol/disulfide exchange experiments, with glutathione as reference redox pair, were performed on the bis(cysteinyl) octapeptides. As the free energy of ring closure of linear peptides consists mainly of the free energy of formation of the disulfide loop with a defined geometry from a statistical ensemble of conformations of the bis(cysteinyl) peptides, the observed differences in the equilibrium constants, although relatively small (within a factor 10), suggest that sequence-dependent information for loop formation is retained in the excised active-site fragments. These inherent redox potentials are, however, significantly affected and/or amplified in the native proteins by the conformational restraints imposed by the "structural domains" on the "functional domains".

Amino Acid Sequence↗
PubMed2004

Interfacial micellar structures from novel amphiphilic star polymers.

An amphiphilic heteroarm star polymer containing 12 alternating hydrophobic/hydrophilic arms of polystyrene (PS) and poly(acrylic acid) (PAA) connected to a well-defined rigid aromatic core was studied at the air-water and the air-solid interfaces. At the air-water interface, the molecules spontaneously form pancakelike micellar aggregates which measure up to several microns in diameter and 5 nm in thickness. Upon reduction of the surface area per molecule to 7 nm2, the two-dimensional micelles merged into a dense monolayer. We suggest that confined phase separation of dissimilar polymer arms occurred upon their segregation on the opposite sides of the rigid disklike aromatic core, forcing the rigid cores to adopt a face-on orientation with respect to the interface. Upon transfer onto solid supports the PS chains face the air-film interface making it completely hydrophobic, and the PAA chains were found to collapse and form a thin flattened underlayer. This study points toward new strategies to create large 2D microstructures with facial amphiphilicity and suggests a profound influence of star molecular architecture on the self-assembly of amphiphiles at the air-water interface.

Acrylic Resins↗
PubMed1999

Elucidation of the structure of an alanine-lacking core tetrasaccharide trisphosphate from the lipopolysaccharide of Pseudomonas aeruginosa mutant H4.

Lipopolysaccharide (LPS) of Pseudomonas aeruginosa rough mutant H4 was isolated by hot water/phenol extraction followed by a modified phenol/chloroform/petroleum ether procedure. Upon SDS/PAGE, the LPS showed a strong major band corresponding to the expected rough-type LPS. Additional faint high molecular-mass bands revealed that the O-chain was present, indicating that the H4 mutant is genetically unstable. Mild acid hydrolysis of the LPS removed lipid A and released a phosphorylated core oligosaccharide that was purified by gel-permeation chromatography and high-performance anion-exchange liquid chromatography. The oligosaccharide contained two residues of L-glycero-D-manno-heptose (Hep) and one residue each of 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and GalNAc. Upon matrix-assisted laser desorption/ionization mass spectroscopy in the negative ion mode, the main fraction expressed a peak for the molecular ion [M-H]- at m/z 1106.41, which was compatible with a carbamoylated, trisphosphorylated tetrasaccharide. The structure was further investigated using one- and two-dimensional homonuclear and heteronuclear correlated NMR spectroscopy at pD 3 and, after borohydride reduction, at pD 9. The NMR data of the two phosphorylated tetrasaccharides recorded at different pD allowed determination of the positions of the three phosphate (P) groups and the carbamoyl group (Cm) thus establishing the following structure of the core oligosaccharide: [equation: see text] Two unusual structural features in the core oligosaccharide of P. aeruginosa were identified for the first time, i.e. the replacement of an amide-linked alanyl group in GalN with an acetyl group and the phosphorylation at position 6 of HepII.

Carbohydrate Conformation↗
PubMed2002

An in vitro methodology for evaluating the mechanical properties of aortic vascular prostheses.

The main problem in the replacement of pathological segments of the aorta with vascular prostheses consists of matching the fluid admittance of the host artery and the graft. This mismatch results from the different compliance between natural and prosthetic vessels and from the plastic dilatation of the prosthesis diameter that occurs after implantation. An experimental procedure was set up for evaluating the mechanical properties of aortic vascular prostheses. An MTS 858 MiniBionix testing machine was equipped with a purposely designed testing apparatus, which allows loading a ring-shaped prosthesis specimen with forces that can be related easily to the transmural pressure acting on the prostheses in vivo. The reference pressure waveforms are simulated from a lumped parameter model of the cardiovascular system. Preliminary tests on 3 different (woven, warp knitted, and carbon-coated warp knitted fabric) aortic prostheses point out a good reproducibility of the results. The fabric strongly affects the circumferential elasticity and the dimensional stability of the graft. Simulation of hypertension promotes larger diameter dilatation and reduction in compliance. Agreement between in vitro and clinical diameter measurements has been assessed for 8 prosthesis samples and found to be adequate. This method is thus a potentially useful means for preclinical evaluation of compliance of vascular prostheses for the purpose of matching to native vessels.

Aged↗
PubMed2004

4-dimensional computed tomography imaging and treatment planning.

In the era of conformal therapy and intensity-modulated therapy, there is an increased desire to raise tumor dose to facilitate improved survival and decrease normal tissue dose to reduce treatment-related complications. Setup accuracy and internal motion limit our ability to reduce margins. Internal motion has both interfraction and intrafraction components, although only the intrafraction component will be addressed here. Intrafraction motion is significant for lung, liver, and pancreatic radiotherapy and to a lesser extent breast and prostate radiotherapy. A method to explicitly account for intrafraction motion is to temporally adjust the treatment beam based on the tumor position with time such that the motion of the radiation beam is synchronized with the tumor motion. This addition of time into the 3-dimensional treatment process is termed 4-dimensional (4D) radiotherapy. Four-dimensional radiotherapy may allow safe clinical target volume-planning target volume margin reduction to achieve the goals of raised tumor dose and decreased normal tissue dose. This article discusses methodology for 4D CT imaging and 4D treatment planning, with some comments on 4D radiation delivery.

Humans↗
PubMed2002

Support vector regression applied to the determination of the developmental age of a Drosophila embryo from its segmentation gene expression patterns.

MOTIVATION: In this paper we address the problem of the determination of developmental age of an embryo from its segmentation gene expression patterns in Drosophila. RESULTS: By applying support vector regression we have developed a fast method for automated staging of an embryo on the basis of its gene expression pattern. Support vector regression is a statistical method for creating regression functions of arbitrary type from a set of training data. The training set is composed of embryos for which the precise developmental age was determined by measuring the degree of membrane invagination. Testing the quality of regression on the training set showed good prediction accuracy. The optimal regression function was then used for the prediction of the gene expression based age of embryos in which the precise age has not been measured by membrane morphology. Moreover, we show that the same accuracy of prediction can be achieved when the dimensionality of the feature vector was reduced by applying factor analysis. The data reduction allowed us to avoid over-fitting and to increase the efficiency of the algorithm.

Aging↗
PubMed2000

Quantitative HRTEM analysis of semiconductor quantum dots

Elastic strains and layer compositions of semiconductor quantum dots are quantified by the measurement of lattice fringe spacings from high-resolution micrographs. Analyses of simulated images, taking thin-specimen relaxation into account by application of finite element simulations, demonstrate that the local slopes of these strain profiles may contain severe artefacts mainly caused by local crystal tilts. Nevertheless, average strain values may be measured with sufficient accuracy and can be used to obtain an estimate on average layer compositions by application of the continuum theory of elasticity when analysing experimental micrographs. Focusing on In(x)Ga1-xAs/GaAs and Ge(x)Si1-x/Si heterostructures, it is demonstrated that elastic strains of nanoscale coherent islands are severely decreased due to an elastic relaxation mechanism compared to fully strained two-dimensional layers. The analysis of self-assembled quantum dots and two-dimensional wetting layers buried by capping layers gives clear evidence for a substantial reduction of the lattice strains compared to the values expected for the nominal layer stoichiometries. This observation originates most presumably from a compositional intermixing during epitaxial growth.

Journal Article↗
PubMed1998

Vertical symphyseal osteotomy.

For the treatment of mild crossbite with increased bigonial distance, the authors performed a vertical symphyseal osteotomy on six patients in the last 3 years. Three of these patients had cleft lip deformities and the others had operations for orthodontic or aesthetic reasons. After exposing the mandible through the buccal mucosal incision, both premolars were extracted with or without conventional segmental osteotomy. The two vertical symphyseal osteotomies were performed with approximately 1 cm between them, and the central part of the mandibular bone was discarded. The bilateral segments of the mandibular body were fixed in the midline using titanium miniplates. Satisfactory results were obtained with a reduction in the size of the mandibular arch, which produced better three-dimensional proportions in the bimaxillary area. No patients had temporomandibular joint problems, however postoperative orthodontics were essential for this type of operation.

Adult↗
PubMed2007

Modeling the cholesteatoma microenvironment: coculture of HaCaT keratinocytes with WS1 fibroblasts induces MMP-2 activation, invasive phenotype, and proteolysis of the extracellular matrix.

BACKGROUND: Increased keratinocyte proliferation, increased keratinocyte migration, elaboration of proteases resulting in proteolysis of the extracellular matrix (ECM), and destruction of surrounding tissues all typify the course of cholesteatoma growth. The contribution of stromal fibroblasts to these behaviors remains relatively unexplored. OBJECTIVES: Our objective for the current studies was to create a simple model with which to study these cholesteatoma behaviors, specifically, cell migration, invasion, and proteolysis of the extracellular matrix as well as the role of fibroblasts in the activated keratinocyte phenotype of cholesteatoma. DESIGN: The authors conducted an in vitro culture model. METHODS: The resulting model consists of activated keratinocytes (HaCaT cells) cocultured with normal dermal fibroblasts (WS1 cells) within a three-dimensional reconstituted ECM. We used a confocal imaging assay and software analysis to quantify total functional proteolysis of the ECM in monotypic and organotypic cocultures. This was accomplished by growing cells on an artificial ECM comprised of Matrigel and DQ-collagen IV. DQ-collagen is a "quenched" fluorescent peptide whose fluorescence is unmasked by proteolytic cleavage. RESULTS: Organotypic cocultures of keratinocytes and fibroblasts exhibited increased cell migration, increased cell invasion, increased matrix metalloproteinase-2 secretion and activation, and increased proteolysis of type IV collagen in three-dimensional ECM. Exposure to NSC27366, inhibitor of the small GTPase, Rac, resulted in reduction in both cell invasion and ECM proteolysis. CONCLUSIONS: Stromal fibroblasts may stimulate the invasive phenotype of keratinocytes, including ECM proteolysis. Increased cell invasion and proteolysis are dependent on the Rac pathway in this model. This simple culture model may help further our understanding of these destructive behaviors in cholesteatoma keratinocytes.

Cell Communication↗