Copine-III interacts with ErbB2 and promotes tumor cell migration.

ErbB2 amplification and overexpression in breast cancer correlates with aggressive disease and poor prognosis. To find novel ErbB2-interacting proteins, we used stable isotope labeling of amino acids in cell culture followed by peptide affinity pull-downs and identified specific binders using relative quantification by mass spectrometry. Copine-III, a member of a Ca(2+)-dependent phospholipid-binding protein family, was identified as binding to phosphorylated Tyr1248 of ErbB2. In breast cancer cells, Copine-III requires Ca(2+) for binding to the plasma membrane, where it interacts with ErbB2 upon receptor stimulation, an interaction that is dependent on receptor activity. Copine-III also binds receptor of activated C kinase 1 and colocalizes with phosphorylated focal adhesion kinase at the leading edge of migrating cells. Importantly, knockdown of Copine-III in T47D breast cancer cells causes a decrease in Src kinase activation and ErbB2-dependent wound healing. Our data suggest that Copine-III is a novel player in the regulation of ErbB2-dependent cancer cell motility. In primary breast tumors, high CPNE3 RNA levels significantly correlate with ERBB2 amplification. Moreover, in an in situ tissue microarray analysis, we detected differential protein expression of Copine-III in normal versus breast, prostate and ovarian tumors, suggesting a more general role for Copine-III in carcinogenesis.

[New condylar head system for temporary condylar reconstruction in ablative tumour surgery]. / Neu entwickeltes Kiefergelenkkopfimplantat zur temporären Kondylus-Rekonstruktion in der ablativen Tumorchirurgie.

BACKGROUND: Based on own retrospective studies a condylar head add-on system for immediate, temporary reconstruction in patients undergoing ablative surgery requiring the removal of the manibular condyle has been developed in cooperation with the Department of Oral and Maxillofacial Surgery of the University of Tennessee, USA, and the Association for the Study of Internal Fixation (AO/ASIF). PURPOSE: The design of the new condylar head add-on system and its use in an anatomical dissection study on a human cadaver are introduced and discussed. DESIGN AND FIRST EXPERIENCES: The condylar replacement is made of commercial pure titanium and is conceived as an add-on system. It consists of a reconstruction plate (2.4 Uni-LOCK-System) und an adaptable condylar head that can be fitted on either side. The offset of the condylar head in a medial direction allows anatomically correct positioning of the implant. The slanted oval head shall provide a large contact area while maintaining function of the mandibular joint. The height-adjustable positioning of the condylar head add-on with four different fixations plates facilitates an intraoperative vertical correction of the condylar head without necessary bending of a new reconstruction plate. A condylar head add-on used on both sides and combined with the frequently used 2.4 Uni-LOCK-plate benefits from reduced storekeeping and turns out to be advantageous from an economic point of view. PERSPECTIVE: An international, prospective multi-center study evaluating the intraoperative applicability of the new condylar head add-on system and its functional as well as aesthetic results during the first two postoperative years has started in September 2006.

[Conductive bone substitute material with variable antibiotic delivery]. / Konduktives Knochenersatzmaterial mit variabler Antibiotikaversetzung.

BACKGROUND: A new bone substitute, consisting of hydroxylapatite and calcium sulphate, was prepared in two formulations and analysed for its mechanical strength and antibiotic elution. MATERIAL AND METHODS: The bone substitute PerOssal has osteoconductive and degradable properties. The material has a built-in capillary structure, which results in an immediate fluid uptake. Antibiotics absorbed to the bone substitute resulted in a prolonged release rate. Mechanical strength was investigated by an unconfined compression test up to failure under both wet and dry conditions for both formulations of the bone substitute. Antibiotic release was analysed microbiologically for two antibiotics, vancomycin and gentamicin, over an elution period of 10 days using the agar diffusion method. RESULTS: The drug release analysis resulted in a prolonged release rate of both antibiotics over 10 days. In vitro the amount of gentamicin and vancomycin eluted at day 10. From one pellet still exceeded the minimal inhibitory concentration of most aetiologically important pathogens. Formulation two of the present bone substitute is significantly harder in both wet and dry conditions when compared to formulation one. Both formulations lose strength in the wet condition relative to their performance in the dry condition. However, formulation two is as hard under wet conditions as formulation one is when dry. CONCLUSION: PerOssal is a suitable new degradable osteoconductive bone substitute that can be loaded with antibiotic solutions, which are released in effective doses over 10 days. The mechanical strength of PerOssal is sufficient to support cancellous bone defects in non-weight-bearing areas or in combination with osteosynthesis.

Antioxidant status of surgical patients receiving TPN with an omega-3-fatty acid-containing lipid emulsion supplemented with alpha-tocopherol.

BACKGROUND: LCT lipid emulsions and even more fish oil-containing lipid emulsions are under debate regarding their tocopherol and PUFA content as well as their effect on the antioxidative status especially in patients with oxidative stress. METHODS: Thirty-three patients undergoing major abdominal surgery were randomly assigned to receive either an alpha-tocopherol-supplemented (562 micromol/l) MCT/LCT/omega-3-acid triglycerides (MLF, 5/4/1 w/w/w, 20%) emulsion or a soybean oil-based LCT emulsion (20%). The TPN regimen continuously provided 1.4 g fat kg bw(-1)d(-1)over 5 days. RESULTS: Plasma antioxidant concentrations were strongly reduced by surgical treatment. Following 5 days of TPN with the MLF emulsion, mean plasma alpha-tocopherol increased by 20.0 micromol/l (1.98 micromol/mmol lipid), while nearly no change was observed in the LCT emulsion group. In both groups, plasma concentrations of all non-supplemented antioxidants (vitamin C, carotenoids, selenium) as well as serum total antioxidant capacity further decreased during TPN. The concentrations of plasma cholesterol oxidation products as a measure of in vivo lipid peroxidation revealed no changes over the TPN period in either group. CONCLUSION: In contrast to the LCT emulsion, administration of the a-tocopherol supplemented MLF lipid emulsion normalized a-tocopherol plasma concentrations. Despite its high long-chain PUFA content, no hint for increased lipid peroxidation was found.

Tissue-specific expression of two genes for sucrose synthase in carrot (Daucus carota L.).

Sucrose synthase, which cleaves sucrose in the presence of uridine diphosphate (UDP) into UDP-glucose and fructose, is thought to be a key determinant of sink strength of heterotrophic plant organs. To determine the roles of the enzyme in carrot, we characterized carrot sucrose synthase at the molecular level. Two genes (Susy*Dc1 and Susy*Dc2) were isolated. The deduced amino acid sequences are 87% identical. However, the sequences upstream of the translation initiation codons are markedly different, as are the expression patterns of the two genes. Susy*Dc2 was exclusively expressed in flowers. Transcripts for Susy*Dc1 were found in stems, in roots at different developmental stages, and in flower buds, flowers and maturing seeds, with the highest levels in strong utilization sinks for sucrose such as growing stems and tap root tips. Expression of Susy*Dc1 was regulated by anaerobiosis but not by sugars or acetate. The carrot sucrose synthase protein is partly membrane-associated and this insoluble form may be directly involved in cellulose biosynthesis. Tap roots of the carrot cultivar used accumulated starch in the vicinity of the vascular bundles, which correlated with high sucrose synthase transcript levels. This finding suggests that soluble sucrose synthase in tap roots channels sucrose towards starch biosynthesis. Starch accumulation appears to be transient and may be involved in sucrose partitioning to developing tap roots.

Two isoforms of plant RAD23 complement a UV-sensitive rad23 mutant in yeast.

DNA repair by nucleotide excision (NER) has been demonstrated in plant cells at the biochemical level but until now none of the molecular components of the plant NER complex has been identified. In this paper, the cloning and characterization of two isoforms of RAD23 from carrot (Daucus carota L.) are reported. It has been suggested that RAD23 in yeast is an assembly factor of the NER complex required for transcription-coupled repair as well as efficient overall genome repair. A functional assay demonstrated that both plant homologues complement the UV-sensitive phenotype of a rad23 deletion mutant of yeast. This result suggests that homologous polypeptides may catalyse NER in plants and yeast and, possibly, by a similar mechanism.

Structural organization and differential expression of carrot beta-fructofuranosidase genes: identification of a gene coding for a flower bud-specific isozyme.

Three genomic clones (Inv*Dc1, Inv*Dc2 and Inv*Dc3) were isolated by using the cDNA for carrot cell wall beta-fructofuranosidase as a probe. The expression patterns of the three genes differed markedly. High levels of Inv*Dc1 transcripts were found in leaves and roots of young carrot, whereas in plants with developing tap roots no transcripts were detected. A high level of mRNA of Inv*Dc1 was also present in suspension-cultured cells. In developing reproductive organs, only low levels of transcripts of Inv*Dc1 were found in flower buds and flowers and none at later stages of development. In contrast, Inv*Dc2 and Inv*Dc3 were not expressed in vegetative plant organs. Invb1*Dc1 was exclusively and strongly expressed in flower buds, and Inv*Dc3 at a very low level in suspension-cultured cells.

cDNA cloning of carrot (Daucus carota) soluble acid beta-fructofuranosidases and comparison with the cell wall isoenzyme.

Carrot (Daucus carota), like most other plants, contains various isoenzymes of acid beta-fructofuranosidase (beta F) (invertase), which either accumulate as soluble polypeptides in the vacuole (isoenzymes I and II) or are ionically bound to the cell wall (extracellular beta F). Using antibodies against isoenzyme I of carrot soluble beta F, we isolated several cDNA clones encoding polypeptides with sequences characteristic of beta Fs, from bacteria, yeast, and plants. The cDNA-derived polypeptide of one of the clones contains all partial peptide sequences of the purified isoenzyme I and thus codes for soluble acid beta F isoenzyme I. A second clone codes for a related polypeptide (63% identity and 77% similarity) with characteristics of isoenzyme II. These two soluble beta Fs, have acidic isoelectric points (3.8 and 5.7, respectively) clearly different from the extracellular enzyme, which has a basic isoelectric point of 9.9. Marked differences among the three nucleotide sequences as well as different hybridization patterns on genomic DNA gel blots prove that these three isoenzymes of carrot acid beta F are encoded by different genes and do not originate from differential splicing of a common gene, as is the case in the yeast Saccharomyces cerevisiae. All three carrot acid beta Fs, are preproenzymes with signal peptides and N-terminal propeptides. A comparison of the sequences of the soluble enzymes with the sequence of the extracellular protein identified C-terminal extensions with short hydrophobic amino acid stretches that may contain the information for vacuolar targeting.

Comparative use of three electrokinetic capillary methods for the determination of drugs in body fluids. Prospects for rapid determination of intoxications.

Three electrokinetic capillary methods, micellar electrokinetic capillary chromatography, capillary zone electrophoresis and capillary isotachophoresis, are shown to be well suited for the rapid screening and confirmation of drugs in serum and urine of patients with medical drug overdoses (intoxications), situations where rapid identification without precise quantification is needed. Patients' samples obtained from the emergency care unit were analysed in an instrument featuring on-column, fast forward-scanning multi-wavelength detection and the data were compared with those obtained by conventional methods. The drugs studied included salicylate, acetaminophen (paracetamol) and antiepileptics. In cases with high drug concentrations, body fluids can be injected directly or may have only to be diluted (urine) or ultrafiltered (serum) prior to analysis, providing results within about 30 min. Thus, electrokinetic capillary methods can be employed for rapid drug screening, provided that instrumentation with a database for peak identification is available.

Strategies for the monitoring of drugs in body fluids by micellar electrokinetic capillary chromatography.

Electrokinetic capillary techniques can exploit numerous separation principles, making them flexible and easily applicable to a variety of separation problems. In recent publications, this emerging technology has been shown to be well suited for monitoring drugs and metabolites in body fluids, including serum, saliva and urine. Most attention has been focused on micellar electrokinetic capillary chromatography (MECC) because it permits the separation and determination of drugs with discrimination being largely based on differences in hydrophobicity. An overview of literature data on the MECC of drugs in body fluids and recent data obtained with antiepileptics in serum and saliva, with model mixtures of illicit drugs, and with extracts from urine specimens that tested positively for opiates and cocaine metabolites are presented. Emphasis is focused on buffer selection and simple sample preparation procedures, including direct injection of body fluids, ultrafiltration and solid-phase extraction.

An enhancer-like sequence within the Xenopus U2 gene promoter facilitates the formation of stable transcription complexes.

Enhancers are eukaryotic promoter elements that increase transcriptional efficiency in a manner relatively independent of their position and orientation with respect to a nearby gene. There is growing evidence that enhancer action is mediated by transacting factors, but the mode of action of these factors is not yet known. We report here on the Xenopus U2 gene promoter, which contains two sequence elements. The distal sequence element increases promoter activity 20-fold by facilitating the formation of stable transcription complexes. A synthetic 14-base-pair (bp) oligonucleotide corresponding to part of the distal sequence element, which shows homology to an immunoglobulin gene promoter element and to both the simian virus 40 (SV40) and the immunoglobulin heavy-chain gene enhancers, stimulates transcription in an orientation-independent manner.

Nuclear exclusion of transcription factor IIIA and the 42s particle transfer RNA-binding protein in Xenopus oocytes: a possible mechanism for gene control?

The intracellular location of 7S and 42S RNP particles in Xenopus oocytes has been determined by immunohistochemistry. Using antibodies directed against the 48-mol-wt protein component of the 42S particle and against transcription factor IIIA, the protein moiety of the 7S particle, we show that these ribonucleoprotein particles are detectable only in the oocyte cytoplasm, being excluded from the nucleus. The mechanism of this nuclear exclusion, and its possible significance in the regulation of 5S RNA gene expression, are discussed.

Intracellular transport of microinjected 5S and small nuclear RNAs.

The mechanism by which some RNAs are segregated in the cell nucleus was analysed by microinjecting 32 P-labelled total RNA from HeLa cells into the cytoplasm of Xenopus oocytes. Small nuclear RNAs (u1, U2, U4, U5 and U6) migrated into the cell nucleus, where they became 30-60 fold more concentrated than in the cytoplasm. Other RNAs, such as tRNA and 7S RNA, remained in the cytoplasm, while 5S RNA became concentrated in the nucleolus. Studies with lupus erythematosus antibodies showed that the migrating RNAs become associated with oocyte RNA-binding proteins.