Vitamin D and Mammographic Findings.

Introduction: Pleiotropic immune-modulatory and anti-proliferative effects of vitamin D and hopes to stop cancerogenesis have led to an increased interest in possible reduction of breast cancer with higher vitamin D levels. Mammographic density is an established risk factor for breast cancer risk, and its association with serum vitamin D is complex, as recent studies have shown. Patients and Methods: In this cross-sectional study, 1103 participants were recruited in the breast diagnostic unit of the Klinikum rechts der Isar, TU Munich. A standardised questionnaire and blood samples for 25-OH-vitamin D were taken on the day of mammography. Histologic results of biopsies in suspicious mammographies were documented. Results: In the 1090 data-sets analysed, vitamin D-deficiency was common among women under 40. Highest vitamin D values were observed in participants aged 60-69 years, but average values for all age cohorts were below 20 ng/ml of vitamin D. 15.6 % of all participants had very low vitamin D values (< 10 ng/ml), 51.3 % were vitamin D-deficient (10-19 ng/ml) and only 5.7 % were above 30 ng/ml, i.e. showed sufficient vitamin D. Patients with malignant results had vitamin D < 10 ng/ml more often (16.9 %; p = 0.61), and only 3.4 % in this group had sufficient vitamin D supply (> 30 ng/ml). There were no significant differences in vitamin D-levels between density groups according to the American College of Radiology (ACR) criteria. Conclusion: Vitamin D values were lower than in comparable US women. Up to now, there is no direct clinical evidence for a relationship between the risk for breast cancer and a specific vitamin D value.

Incidence of axillary recurrence in 113 sentinel node negative breast cancer patients: a 3-year follow-up study.

BACKGROUNDS/AIMS: This study evaluates the 3-year follow-up period and recurrence rate in patients with a negative sentinel node biopsy (SNB) without an additional axillary dissection (ALND). METHODS: Between January 2000 and March 2002, 197 patients with an invasive breast cancer and clinically negative axillary nodes underwent a sentinel node biopsy. One hundred and thirteen patients were included in our study. The follow-up consisted of clinical examination every 3 months in the first year, followed by every 6 months after the first year. A mammography was obtained annually. Attention was paid to loco-regional recurrence, including axillary recurrence, and distant metastases. RESULTS: The mean duration of follow-up was 37.5 months (range 24-54). In this period, one patient was diagnosed with an axillary recurrence and one patient developed a supraclavicular lymph node metastasis. Two patients developed a second primary breast cancer in the contralateral breast. No patients were diagnosed with distant metastasis. CONCLUSION: These 3 year follow-up results suggest that SNB is a procedure with a low clinical recurrence rate, which can replace, when strict criteria are met, ALND if the sentinel node is negative.

Optimised fermentation strategy for 13C/15N recombinant protein labelling in Escherichia coli for NMR-structure analysis.

A widely applicable cultivation strategy, which reduces the costs of expensive isotopes, is designed for maximal (98-100%) incorporation of [13C] and [15N] into labelled recombinant protein expressed in Escherichia coli, allowing better assignment of the resonances for NMR studies. Isotope labelling of the culture was performed throughout the complete process, starting from preculture. Sufficient biomass is first generated in a batch phase. Upon consumption of glucose, identified by a sharp drop of on-line monitored oxygen consumption, expression is induced and cultivation is continued under glucose-limited conditions as fed-batch process. Thereby a quantitative utilisation of the most expensive component [13C]-glucose is achieved, while the approximate amount of the [15N]-ammonium chloride to be incorporated is calculated from the scheduled biomass. The usefulness of the strategy is demonstrated with production of uniformly [13C/15N]-labelled tryparedoxin of Crithidia fasciculata. Ideal isotope incorporation and product quality is documented by MALDI-TOF mass spectrometry and two- and three-dimensional NMR spectra.

Structures of tryparedoxins revealing interaction with trypanothione.

Tryparedoxins (TXNs) catalyse the reduction of peroxiredoxin-type peroxidases by the bis-glutathionyl derivative of spermidine, trypanothione, and are relevant to hydroperoxide detoxification and virulence of trypanosomes. The 3D-structures of the following tryparedoxins are presented: authentic tryparedoxin1 of Crithidia fasciculata, CfTXN1; the his-tagged recombinant protein, CfTXN1H6; reduced and oxidised CfTXN2, and an alternative substrate derivative of the mutein CfTXN2H6-Cys44Ser. Cys41 (Cys40 in TXN1) of the active site motif 40-WCPPCR-45 proved to be the only solvent-exposed redox active residue in CfTXN2. In reduced TXNs, its nucleophilicity is increased by a network of hydrogen bonds. In oxidised TXNs it can be attacked by the thiol of the 1N-glutathionyl residue of trypanothione, as evidenced by the structure of 1N-glutathionylspermidine-derivatised CfTXN2H6-Cys44Ser. Modelling suggests Arg45 (44), Glu73 (72), the Ile110 (109) cis-Pro111 (110)-bond and Arg129 (128) to be involved in the binding of trypanothione to CfTXN2 (CfTXN1). The model of TXN-substrate interaction is consistent with functional characteristics of known and newly designed muteins (CfTXN2H6-Arg129Asp and Glu73Arg) and the 1N-glutathionyl-spermidine binding in the CfTXN2H6-Cys44Ser structure.

Risk, severity and predictors of physical and psychological morbidity after axillary lymph node dissection for breast cancer.

The aim of this study was to investigate the nature and severity of the arm complaints among breast cancer patients after axillary lymph node dissection (ALND) and to study the effects of this treatment-related morbidity on daily life and well-being. 400 women, who underwent ALND as part of breast cancer surgery, filled out a treatment-specific quality of life questionnaire. The mean time since ALND was 4.7 years (range 0.3-28 years). More than 20% of patients reported pain, numbness, or loss of strength and 9% reported severe oedema. None of the complaints appeared to diminish over time. Irradiation of the axilla and supraclavicular irradiation were associated with a 3.57-fold higher risk of oedema (odds ratio (OR) 3.57; 95% confidence interval (CI) 1.66-7.69) causing many patients to give up leisure activities or sport. Women who underwent irradiation of the breast or chest wall more often reported to have a sensitive scar than women who did not receive radiotherapy. Women <45 years of age had an approximately 6 times higher risk of numbness of the arm (OR 6.49; 95% CI 2.58-16.38) compared with those > or = 65 years of age; they also encountered more problems doing their household chores. The results of the present study support the introduction of less invasive techniques for the staging of the axilla, sentinel node biopsy being the most promising.

Enrichment of hydrophobic proteins via Triton X-114 phase partitioning and hydroxyapatite column chromatography for mass spectrometry.

Membrane proteins are the starting point of several signal transduction pathways. Therefore, the separation and identification of these proteins are of great interest in proteome analysis. However, the specific properties of membrane proteins seriously impede their analysis. We present an effective and highly reproducible method for the two-dimensional separation of extremely hydrophobic proteins and demonstrate the advantages of special preseparation procedures for the identification of proteins which have very similar Mr and p/. Using the example of the integral membrane protein very low density lipoprotein (VLDL) receptor (NCBI Acc. # 1730111) and the soluble heat shock protein (HSP) 90 (NCBI Acc. # 386786) we present the applicability of a phase-separation system with Triton X-114. Using matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) of the protein spots after 2-D separation of the hydrophilic and the strongly hydrophobic protein fraction of human endothelial cells (ECV cell line), we were able to distinguish both proteins.

Dual function of the selenoprotein PHGPx during sperm maturation.

The selenoprotein phospholipid hydroperoxide glutathione peroxidase (PHGPx) changes its physical characteristics and biological functions during sperm maturation. PHGPx exists as a soluble peroxidase in spermatids but persists in mature spermatozoa as an enzymatically inactive, oxidatively cross-linked, insoluble protein. In the midpiece of mature spermatozoa, PHGPx protein represents at least 50 percent of the capsule material that embeds the helix of mitochondria. The role of PHGPx as a structural protein may explain the mechanical instability of the mitochondrial midpiece that is observed in selenium deficiency.

Testosterone mediates expression of the selenoprotein PHGPx by induction of spermatogenesis and not by direct transcriptional gene activation.

Selenium deficiency is known to be associated with male infertility, and the selenoprotein PHGPx has been shown to increase in rat testis after puberty and to depend on gonadotropin stimulation in hypophysectomized rats [Roveri et al. (1992) J. Biol. Chem. 267, 6142 6146]. Exposure of decapsulated whole testis, however, failed to reveal any transcriptional activation or inhibition of the PHGPx gene by testosterone, human chorionic gonadotropin, or forskolin. Nevertheless, it was verified that the specific activity of PHGPx in testis, but not of cGPx, correlated with sexual maturation. Leydig cell destruction in vivo by ethane dimethane sulfonate (EDS) resulted in a delayed decrease in PHGPx activity and mRNA that could be completely prevented by testosterone substitution. cGPx transiently increased upon EDS treatment, probably as a result of reactive macrophage augmentation. In situ mRNA hybridization studies demonstrated an uncharacteristic low level of cGPx transcription in testis, whereas PHGPx mRNA was abundantly and preferentially expressed in round spermatids. The data show that the age or gonadotropin-dependent expression of PHGPx in testis does not result from direct transcriptional gene activation by testosterone, but is due to differentiation stage-specific expression in late spermatids, which are under the control of Leydig cell-derived testosterone. The striking burst of PHGPx expression at the transition of round to elongated spermatids suggests an involvement of this selenoprotein in sperm maturation.

The Escherichia coli pgpB gene encodes for a diacylglycerol pyrophosphate phosphatase activity.

We provided genetic and biochemical evidence that supported the conclusion that the product of pgpB gene of Escherichia coli exhibited diacylglycerol pyrophosphate (DGPP) phosphatase activity. DGPP phosphatase activity was absent in pgpB mutant cells and was expressed at high levels in cells carrying the wild-type pgpB gene on a runaway replication plasmid. The pgpB mutant has been primarily characterized by a defect in phosphatidate (PA) phosphatase activity and also exhibits defects in lyso-PA phosphatase and phosphatidylglycerophosphate phosphatase activities. The defective PA phosphatase in the pgpB mutant was shown to be a Mg2+-independent PA phosphatase activity of the DGPP phosphatase enzyme. We characterized DGPP phosphatase activity in membranes from cells overproducing the pgpB gene product. DGPP phosphatase catalyzed the dephosphorylation of the beta phosphate of DGPP to form PA followed by the dephosphorylation of PA to form diacylglycerol. The specificity constant (Vmax/Km) for DGPP was 9.3-fold greater than that for PA. The pH optimum for the DGPP phosphatase reaction was 6. 5. Activity was independent of a divalent cation requirement, was potently inhibited by Mn2+ ions, and was insensitive to inhibition by N-ethylmaleimide. Pure DGPP phosphatase from Saccharomyces cerevisiae was shown to be similar to the E. coli DGPP phosphatase in its ability to utilize lyso-PA and phosphatidylglycerophosphate as substrates in vitro.

Purification and characterization of diacylglycerol pyrophosphate phosphatase from Saccharomyces cerevisiae.

Diacylglycerol pyrophosphate (DGPP) phosphatase is a novel membrane-associated enzyme that catalyzes the dephosphorylation of the beta phosphate of DGPP to yield phosphatidate and Pi. DGPP phosphatase was purified 33,333-fold from Saccharomyces cerevisiae by a procedure that included Triton X-100 solubilization of microsomal membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, and Mono Q. The procedure resulted in the isolation of an apparent homogeneous protein with a subunit molecular mass of 34 kDa. DGPP phosphatase activity was associated with the 34-kDa protein. DGPP phosphatase had a broad pH optimum between 6.0 and 8.5 and was dependent on Triton X-100 for maximum activity. The enzyme was inhibited by divalent cations, NaF, and pyrophosphate and was relatively insensitive to thioreactive agents. The turnover number (molecular activity) for the enzyme was 5.8 x 10(3) min-1 at pH 6.5 and 30 degrees C. DGPP phosphatase exhibited typical saturation kinetics with respect to DGPP (Km = 0.55 mol %). The Km value for DGPP was 3-fold greater than its cellular concentration (0.18 mol %). DGPP phosphatase also catalyzed the dephosphorylation of phosphatidate, but this dephosphorylation was subsequent to the dephosphorylation of the beta phosphate of DGPP. The dependence of activity on phosphatidate (Km = 2.2 mol %) was cooperative (Hill number = 2.0). DGPP was the preferred substrate for the enzyme with a specificity constant (Vmax/Km) 10-fold greater than that for phosphatidate. In addition, DGPP potently inhibited (Ki = 0.35 mol %) the dephosphorylation of phosphatidate by a competitive mechanism whereas phosphatidate did not inhibit the dephosphorylation of DGPP. DGPP was neither a substrate nor an inhibitor of pure phosphatidate phosphatase from S. cerevisiae. DGPP was synthesized from phosphatidate via the phosphatidate kinase reaction.

Phosphatidate Kinase, A Novel Enzyme in Phospholipid Metabolism (Characterization of the Enzyme from Suspension-Cultured Catharanthus roseus Cells).

Phosphatidate kinase (adenosine 5[prime]-triphosphate:phosphatidic acid phosphotransferase), a novel enzyme of phospholipid metabolism, was detected recently in the plasma membranes of suspension-cultured Catharanthus roseus cells and purified (J.B. Wissing, H. Behrbohm [1993] Plant Physiol 102: 1243-1249). In the present work the properties of phosphatidate kinase are described. The enzyme showed a pH optimum of 6.1 and an isoelectric point of 4.8, and was rather stable in the presence of its substrates. Although the kinase accepted both ATP and GTP, with Km values of about 12 and 18 [mu]M, respectively, the only lipid substrate was phosphatidic acid; neither lysophosphatidic acid nor any other lipid tested was phosphorylated. With 32P- and 14C-labeled diacylglycerol pyrophosphate, the product of the enzyme, it was shown that the kinase catalyzes a reversible reaction. The activity of the extracted enzyme depended on the presence of surfactants such as Triton X-100 or [beta]-octylglucoside, whereas deoxycholate was strongly inhibitory. Kinetic analysis with Triton X-100/phosphatidate mixed micelles performed according to the "surface dilution" kinetic model showed saturation kinetics with respect to both bulk and surface concentration of phosphatidate. The interfacial Michaelis constant for phosphatidate was determined as 0.6 mol %.

Phosphatidate Kinase, a Novel Enzyme in Phospholipid Metabolism (Purification, Subcellular Localization, and Occurrence in the Plant Kingdom).

Microsomal membranes from suspension-cultured Catharanthus roseus cells possess an enzymic activity that catalyzes the ATP-dependent phosphorylation of phosphatidic acid (PA) to form diacylglycerol pyrophosphate (H. Behrbohm, J.B. Wissing [1993] FEBS Lett 315: 95-99). This enzyme activity, PA kinase, was purified and characterized. Plasma membranes, obtained from C. roseus microsomes by aqueous two-phase partitioning, were extracted, and PA kinase was purified 3200-fold by applying different chromatographic steps that resulted in a specific activity of about 10 [mu]mol min-1 mg-1. Sodium dodecyl sulfate-gel electrophoresis of the fractions obtained from the final chromatographic step revealed a 39-kD protein that correlated with the enzyme activity; PA kinase activity could be eluted from this protein band. Subcellular localization, investigated with C. roseus cells, showed that the activity was confined to membrane fractions, and at least 80% was associated with plasma membranes. The data revealed the same distribution within the cellular membranes of PA kinase as reported for diacylglycerol kinase, which is a typical plasma membrane-located enzyme. Furthermore, PA kinase activity was detected in the calli of 16 different plant species and in the different organs of C. roseus plants and obviously occurs ubiquitously in the plant kingdom.

Extensive salivary contamination due to concurrent use of chewing tobacco during I-131 radioablative therapy.

Although multiple authors have reported on various causes of external I-131 contamination after administration of radioiodine, to the authors' knowledge there have been no reported cases of external contamination from salivary secretions due to expectoration of chewing tobacco. A case of extensive I-131 contamination of a hospital room, furniture, and plumbing due to indiscriminate use of chewing tobacco during an inpatient I-131 radioablative therapy is presented.

Diacylglycerol pyrophosphate, a novel phospholipid compound.

In studies on lipid kinase activities of microsomal membranes from cultured plant cells a new, hitherto unknown, lipid kinase product was detected. The new phospholipid, labeled by [gamma-32P]ATP, could be separated from known phospholipid species by thin layer chromatography using different solvent mixtures. After partial purification of the related enzyme activity, the substrate of the unknown lipid kinase was elucidated as phosphatidic acid. With authentic phosphatidic acid and partially purified enzyme, the lipid kinase product was prepared in mg quantities and its structure was determined by mass spectrometry and NMR analyses as diacylglycerol pyrophosphate, a hitherto unknown phospholipid. The possible physiological role of this novel phospholipid metabolite is discussed.

Diacylglycerol kinase from suspension cultured plant cells : characterization and subcellular localization.

Diacylglycerol kinase (adenosine 5'-triphosphate:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107), purified from suspension cultured Catharanthus roseus cells (J Wissing, S Heim, KG Wagner [1989] Plant Physiol 90: 1546-1551), was further characterized and its subcellular location was investigated. The enzyme revealed a complex dependency on lipids and surfactants; its activity was stimulated by certain phospholipids, with phosphatidylinositol and phosphatidylglycerol as the most effective species, and by deoxycholate. In the presence of Triton X-100, used for its purification, a biphasic dependency upon diacylglycerol was observed and the apparent Michaelis constant values for diacylglycerol decreased with decreasing Triton concentration. The enzyme accepted both adenosine 5'-triphosphate and guanosine 5'-triphosphate as substrate and showed rather low apparent inhibition constant values for all nucleoside diphosphates tested. Diacylglycerol kinase is an intrinsic membrane protein and no activity was found in the cytosol. An investigation of different cellular membrane fractions confirmed its location in the plasma membrane.

The posterior antiglide plate for fixation of fractures of the lateral malleolus.

Anatomical reduction and internal fixation of displaced lateral malleolar fractures are the cornerstone of the operative treatment of ankle fractures. The classical method of fixation is the application of one-third tubular plates laterally to the distal fibula, a technique, however, that has several disadvantages. In exceptional cases and under special circumstances we prefer a dorsal approach with the use of an antiglide plate. Indications, technique and experiences are discussed.

[Tension band osteosynthesis of resorbable material]. / Die Zuggurtungsosteosynthese aus resorbierbarem Material.

Tension-band osteosynthesis is a simple and well-known procedure applied in the surgical treatment of fractures. This type of osteosynthesis does nonetheless have some disadvantages, e.g., irritation of the skin and fistulization at the point where the metal cerclage bends, that can make removal necessary mostly under full anaesthesia. Since 1987 we have replaced the metal cerclage in 36 tension-band osteosyntheses (21 fractures of the medial malleolus, 6 lateral malleolar fractures, 4 patella, 4 olecranon and 1 distal clavicular fracture) with the resorbable suture material Vicryl No 2. Our results were good: there have been no skin problems at all and there are several advantages.