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Protein kinase D2 (PKD2), a member of the PKD family of serine/threonine kinases, is localized in various subcellular compartments including the nucleus where the kinase accumulates upon activation of G-protein-coupled receptors. We define three critical post-translational modifications required for nuclear accumulation of PKD2 in response to activation of the CCK2 receptor (CCK2R): phosphorylation at Ser706 and Ser710 within the activation loop by PKC eta leading to catalytic activity and phosphorylation at Ser244 within the zinc-finger domain, which is crucial for blocking nuclear export of active PKD2 by preventing its interaction with the Crm-1 export machinery. We identify CK1delta and epsilon as upstream activated kinases by CCK2R that phosphorylate PKD2 at Ser244. Moreover, nuclear accumulation of active PKD2 is a prerequisite for efficient phosphorylation of its nuclear substrate, HDAC7. Only nuclear, active PKD2 mediates CCK2R-induced HDAC7 phosphorylation and Nur77 expression. Thus, we define a novel, compartment-specific signal transduction pathway downstream of CCK2R that phosphorylates PKD2 at three specific sites, results in nuclear accumulation of the active kinase and culminates in efficient phosphorylation of nuclear PKD2 substrates in human gastric cancer cells.

In the present study the structures of two glycopeptides (G1 and G1′), isolated from FU RvH 1 -b and two glycopeptides (G2 and G3), isolated from the structural subunit RvH 1 of Rapana venosa hemocyanin, were determined. To structurally characterize the site-specific carbohydrate heterogeneity and binding site of the N-linked glycopeptide(s), a combination of capillary reversed-phase chromatography and ion trap mass spectrometry was used. The amino acid sequences of glycopeptides G1 and G1′ determined by Edman degradation and MS/MS sequencing demonstrated that the oligosaccharides are linked to N-glycosylation sites. Two peptides (a glycosylated (G1) and non-glycosylated one) were identified in this fraction and no linkage sites were observed in the latter one. Based on the sequencing of the glycosylated fractions G1, G1′, G2 and G3, the carbohydrate structure Man( α 1–6)Man( α 1–3)Man( β 1–4)GlcNAc( β 1–4)[Fuc( α 1–6)]GlcNAc-R could be identified for glycopeptides G1 and G3, and only the typical core structure Man( α 1–6)Man( α 1–3)Man( β 1–4)GlcNAc( β 1–4)GlcNAc-R was found for G1′ and G2. The Fuc residue found in glycopeptides G1 and G3 is attached to N -acetyl-glucosamine of the carbohydrate core, as often found in other glycoproteins.

A derivative (1) of the immunopotentiating 28-peptide thymosin alpha1 has been especially designed, so that it can be (99m)Tc-radiolabelled, and synthesized following the Fmoc solid-phase peptide synthesis approach. Derivative 1 contains the N-terminal fragment Talpha1[1-14] as a bioactive segment, at the C-terminus of which a (99m)Tc-chelating moiety consisting of N(alpha),N(alpha)-dimethylglycine, serine and cysteine is linked through the N(epsilon)-amino group of a 'bifunctional' lysine residue; the latter is indirectly anchored on the solid-phase peptide synthesis resin through 6-aminocaproic acid (dmGSCK{N(epsilon)-Talpha1[1-14]}Aca). Synthetic derivative 1 was obtained at high overall yield (approximately 35%) and purity (>95%) and shown to be efficiently radiolabelled with (99m)Tc, thus resulting in the first, to our knowledge, so far reported (99m)Tc-radiolabelled derivative of thymosin alpha1, which may be eventually used as a specific molecular tool for the in vitro/in vivo study of the mode of action of the parent bioactive peptide.

Vertebrate metallothioneins are found to contain Zn(II) and variable amounts of Cu(I), in vivo, and are believed to be important for d10-metal control. To date, structural information is available for the Zn(II) and Cd(II) forms, but not for the Cu(I) or mixed metal forms. Cu(I) binding to metallothionein-1 has been investigated by circular dichroism, luminescence and 1H NMR using two synthetic fragments representing the alpha- and the beta-domain. The 1H NMR data and thus the structures of Zn4alpha metallothionein (MT)-1 and Zn3betaMT-1 were essentially the same as those already published for the corresponding domains of native Cd7MT-1. Cu(I) titration of the Zn(II)-reconstituted domains provided clear evidence of stable polypeptide folds of the three Cu(I)-containing alpha- and the four Cu(I)-containing beta-domains. The solution structures of these two species are grossly different from the structures of the starting Zn(II) complexes. Further addition of Cu(I) to the two single domains led to the loss of defined domain structures. Upon mixing of the separately prepared aqueous three and four Cu(I) loaded alpha- and beta-domains, no interaction was seen between the two species. There was neither any indication for a net transfer of Cu(I) between the two domains nor for the formation of one large single Cu(I) cluster involving both domains.

Proteasomal proteolysis relies on the activity of six catalytically active proteasomal subunits (beta1, beta2, beta5, beta1i, beta2i and beta5i). Applying a functional proteomics approach, we used a recently developed activity-based, cell-permeable proteasome-specific probe that for the first time allows differential visualization of individual active proteasomal subunits in intact primary cells. In primary leukemia samples, we observed remarkable variability in the amounts of active beta1/1i-, beta2/2i- and beta5/5i-type of subunits, contrasting with their constant protein expression. Bortezomib inhibited beta5- and beta1-type, but to a lesser extend beta2-type of subunits in live primary cells in vitro and in vivo. When we adapted the bortezomib-sensitive human acute myeloid leukemia cell line HL-60 to bortezomib 40 nM (HL-60a), proteasomal activity profiling revealed an upregulation of active subunits, and residual beta1/beta5-type of activity could be visualized in the presence of bortezomib 20 nM, in contrast to control cells. In a panel of cell lines from hematologic malignancies, the ratio between beta2-type and (beta1 + beta5)-type of active proteasomal polypeptides mirrored different degrees of bortezomib sensitivity. We thus conclude that the proteasomal activity profile varies in primary leukemia cells, and that the pattern of proteasomal subunit activity influences the sensitivity of hematologic malignancies toward bortezomib.

The protein pattern of healthy human eccrine sweat was investigated and 10 major proteins were detected from which apolipoprotein D, lipophilin B, and cathepsin D (CatD) were identified for the first time in human eccrine sweat. We focused our studies on the function of the aspartate protease CatD in sweat. In vitro digestion experiments using a specific fluorescent CatD substrate showed that CatD is enzymatically active in human sweat. To identify potential substrates of CatD in human eccrine sweat LL-37 and DCD-1L, two antimicrobial peptides present in sweat, were digested in vitro with purified CatD. LL-37 was not significantly digested by CatD, whereas DCD-1L was cleaved between Leu(44) and Asp(45) and between Leu(29) and Glu(30) almost completely. The DCD-1L-derived peptides generated in vitro by CatD were also found in vivo in human sweat as determined by surface-enhanced laser desorption/ionization (SELDI) mass spectrometry. Furthermore, besides the CatD-processed peptides we identified additionally DCD-1L-derived peptides that are generated upon cleavage with a 1,10-phenanthroline-sensitive carboxypeptidase and an endoprotease. Taken together, proteolytic processing generates 12 DCD-1L-derived peptides. To elucidate the functional significance of postsecretory processing the antimicrobial activity of three CatD-processed DCD-1L peptides was tested. Whereas two of these peptides showed no activity against Gram-positive and Gram-negative bacteria, one DCD-1L-derived peptide showed an even higher activity against Escherichia coli than DCD-1L. Functional analysis indicated that proteolytic processing of DCD-1L by CatD in human sweat modulates the innate immune defense of human skin.

Prothymosin alpha (proTalpha) is a 109 amino acid long polypeptide presenting distinct immunoenhancing activity in vitro and in vivo. Recent reports suggest that in apoptotic cells, proTalpha is cleaved by caspases at its carboxy(C)-terminus generating potentially bioactive fragments. In this study, we identified the peptide segment of proTalpha presenting maximum immunomodulatory activity. Calf thymus proTalpha was trypsinised, and the five fragments produced (spanning residues 1-14, 21-30, 31-87, 89-102 and 103-109) were tested for their ability to stimulate healthy donor- and cancer patient-derived peripheral blood mononuclear cell (PBMC) proliferation in autologous mixed lymphocyte reaction (AMLR), natural killer and lymphokine-activated killer cell activity, intracellular production of perforin, upregulation of adhesion molecules and CD25 expression. ProTalpha(89-102) and proTalpha(103-109) significantly fortified healthy donor-lymphocytes' immune responses to levels comparable to those induced by intact proTalpha. These effects were more pronounced in cancer patients, where peptides proTalpha(89-102) and proTalpha(103-109) partly, however significantly, restored the depressed AMLR and cytolytic ability of PBMC, by simulating the biological activity exerted by intact proTalpha. ProTalpha(1-14), proTalpha(21-30) and proTalpha(31-87) marginally upregulated lymphocyte activation. This is the first report showing that proTalpha's immunomodulating activity can be substituted by its C-terminal peptide(s). Whether generation and externalization of such immunoactive proTalpha fragments occurs in vivo, needs further investigation. However, if these peptides can trigger immune responses, they may eventually be used therapeutically to improve some PBMC functions of cancer patients.

Scorpion venoms are among the most widely known source of peptidyl neurotoxins used for callipering different ion channels, e.g., for Na(+), K(+), Ca(+) or Cl(-). An alpha-toxin (Bs-Tx28) has been purified from the venom of scorpion Buthus sindicus, a common yellow scorpion of Sindh, Pakistan. The primary structure of Bs-Tx28 was established using a combination of MALDI-TOF-MS, LC-ESI-MS, and automated Edman degradation analysis. Bs-Tx28 consists of 65 amino acid residues (7274.3+/-2Da), including eight cysteine residues, and shows very high sequence identity (82-94%) with other long-chain alpha-neurotoxins, active against receptor site-3 of mammalian (e.g., Lqq-IV and Lqh-IV from scorpions Leiurus sp.) and insect (e.g., BJalpha-IT and Od-1 from Buthotus judaicus and Odonthobuthus doriae, respectively) voltage-gated Na(+) channels. Multiple sequence alignment and phylogenetic analysis of Bs-Tx28 with other known alpha- and alpha-like toxins suggests the presence of a new and separate subfamily of scorpion alpha-toxins. Bs-Tx28 which is weakly active in both, mammals and insects (LD(50) 0.088 and 14.3microg/g, respectively), shows strong induction of the rat afferent nerve discharge in a dose-dependent fashion (EC(50)=0.01microg/mL) which was completely abolished in the presence of tetrodotoxin suggesting the binding of Bs-Tx28 to the TTX-sensitive Na(+)-channel. Three-dimensional structural features of Bs-Tx28, established by homology modeling, were compared with other known classical alpha-mammal (AaH-II), alpha-insect (Lqh-alphaIT), and alpha-like (BmK-M4) toxins and revealed subtle variations in the Nt-, Core-, and RT-CT-domains (functional domains) which constitute a "necklace-like" structure differing significantly in all alpha-toxin subfamilies. On the other hand, a high level of conservation has been observed in the conserved hydrophobic surface with the only substitution of W43 (Y43/42) and an additional hydrophobic character at position F40 (L40/A/V/G39), as compared to the other mentioned alpha-toxins. Despite major differences within the primary structure and activities of Bs-Tx28, it shares a common structural and functional motif (e.g., transRT-farCT) within the RT-CT domain which is characteristic of scorpion alpha-mammal toxins.

The search for novel molecular markers of tumor invasion is vital if strategies are to become more effective in the diagnostic and prognostic management of transitional cell carcinoma of the bladder. Up to 50% of tumors detected at stage 1 (pT1) progress to a higher grade even after endoscopic surgical resection, and there are currently no protein markers of this aggressive, invasive phenotype. We have combined SELDI-TOF-MS, ClinProt magnetic bead enrichment, Nano-LC-ESI-ion trap tandem mass spectrometry and immunohistochemical analysis to the study of 12 invasive bladder cancer tissue biopsies paired with normal bladder tissue samples obtained from the same patients for the definition and identification of proteins up-regulated in the tumors. We report the inflammation-associated calcium binding protein S100A8 (MRP-8, calgranulin A) to be highly expressed in tumor cells in contrast to normal urothelium in 50% of the samples, as well as two unidentified protein markers at 5.75 and 6.89 kDa that were differentially detected in 9/12 and 10/12 tumor samples, respectively. These new markers, when fully characterized, may contribute to new target proteins for the prediction of aggressive, invasive bladder tumors.

In this study, we describe a differential mass spectrometric technique for the immuno-proteomic analysis of the major histocompatibility complex (MHC) peptides of a renal cell carcinoma (RCC) biopsy compared with the healthy kidney tissue of the same patient after nephrectomy. Using a stable isotope labeling approach, we could directly compare and relatively quantify 43 MHC-peptide pairs, most of which were present in similar proportions on both normal kidney and tumor. Significantly, two dominant peptides of monoisotopic masses ([M+H](+)) 973.43 u and 967.59 u, respectively, were found exclusively in the tumor sample. One of these was identified as originating from heme oxygenase-1 (HO-1), a protein involved in induction of apoptosis resistance, immuno-suppression and neoangiogenesis and reported to be up-regulated in various cancer types. Moreover, the corresponding synthetic HO-1-derived peptide was shown to be immunogenic in vitro by generation of CD8+ T cell lines with peptide-specific cytolytic activity. Thus, this peptide is an example of a differentially identified T cell epitope that could be considered as a target for immunotherapy.

Variational data assimilation systems require the specification of the covariances of background and observation errors. Although the specification of the background-error covariances has been the subject of intense research, current operational data assimilation systems still rely on essentially static and thus flow-independent background-error covariances. At least theoretically, it is possible to use flow-dependent background-error covariances in four-dimensional variational data assimilation (4DVAR) through exploiting the connection between variational data assimilation and estimation theory. This paper reports on investigations concerning the impact of flow-dependent background-error covariances in an idealized 4DVAR system that, based on quasigeostrophic dynamics, assimilates artificial observations. The main emphasis is placed on quantifying the improvement in analysis quality that is achievable in 4DVAR through the use of flow-dependent background-error covariances. Flow dependence is achieved through dynamical error-covariance evolution based on singular vectors in a reduced-rank approach, referred to as reduced-rank Kalman filter (RRKF). The RRKF yields partly dynamic background-error covariances through blending static and dynamic information, where the dynamic information is obtained from error evolution in a subspace of dimension k (defined here through the singular vectors) that may be small compared to the dimension of the model’s phase space n, which is equal to 1449 in the system investigated here. The results show that the use of flow-dependent background-error covariances based on the RRKF leads to improved analyses compared to a system using static background-error statistics. That latter system uses static background-error covariances that are carefully tuned given the model dynamics and the observational information available. It is also shown that the performance of the RRKF approaches the performance of the extended Kalman filter, as k approaches n. Results therefore support the hypothesis that significant analysis improvement is possible through the use of flow-dependent background-error covariances given that a sufficiently large number (here on the order of n/10) of singular vectors is used.

Insulin receptor substrate-1 (IRS-1) was recently identified as a novel upstream substrate for the insulin-activated protein kinase C (PKC)-zeta. This interaction down-regulates insulin signal transduction under hyper-insulinemic conditions. To clarify the molecular mechanism of this feedback loop, we sought to identify the PKC-zeta phosphorylation sites of IRS-1 and to investigate their biological significance. Upon incubation of recombinant IRS-1 fragments with PKC-zeta, we identified Ser(318) of rat IRS-1 (Ser(323) in human IRS-1) as the major in vitro phosphorylation site (confirmed by mutation of Ser(318) to alanine). To monitor phosphorylation of Ser(318) in cellular extracts, we prepared a polyclonal phosphosite-specific antibody. The biological significance was studied in baby hamster kidney cells stably expressing the insulin receptor (BHK(IR)). Using the phospho-Ser(318)-specific antibody we observed that insulin stimulates phosphorylation of Ser(318) in IRS-1, which is mediated, at least partially, by PKC-zeta. Moreover, we found that the previously described insulin-stimulated, PKC-zeta-mediated inhibition of the interaction of IRS-1 with the insulin receptor and the reduced tyrosine phosphorylation of IRS-1 was abrogated by mutation of IRS-1 Ser(318) to alanine. These results, generated in BHK(IR) cells, suggest that phosphorylation of Ser(318) by PKC-zeta might contribute to the inhibitory effect of prolonged hyperinsulinemia on IRS-1 function.

PURPOSE To assess safety and efficacy of CT-guided brachytherapy of liver malignancies. PATIENTS AND METHODS 21 patients with 21 liver malignancies (19 metastases, two primary liver tumors) were treated with interstitial CT-guided brachytherapy applying a (192)Ir source. In all patients, the use of image-guided thermal tumor ablation such as by radiofrequency or laser-induced thermotherapy (LITT) was impeded either by tumor size > or = 5 cm in seven, adjacent portal or hepatic vein in ten, or adjacent bile duct bifurcation in four patients. Dosimetry was performed using three-dimensional CT data sets acquired after CT-guided positioning of the brachytherapy catheters. RESULTS The mean tumor diameter was 4.6 cm (2.5-11 cm). The mean minimal tumor dose inside the tumor margin amounted to 17 Gy (12-20 Gy). The proportion of the liver parenchyma exposed to > 5 Gy was 18% (5-39%) of total liver parenchyma minus tumor volume. Nausea and vomiting were observed in six patients after brachytherapy (28%). One patient demonstrated obstructive jaundice due to tumor edema after irradiation of a metastasis adjacent to the bile duct bifurcation. We commonly encountered asymptomatic increases of liver enzymes. Local control rates after 6 and 12 months were 87% and 70%, respectively. CONCLUSION CT-guided brachytherapy is safe and effective. This technique displays broader indications compared to image-guided thermal ablation by radiofrequency or LITT with respect to tumor size or localization.

The asparagine-specific endoprotease (AEP) controls lysosomal processing of the potential autoantigen myelin basic protein (MBP) by human B lymphoblastoid cells, a feature implicated in the immunopathogenesis of multiple sclerosis. In this study, we demonstrate that freshly isolated human B lymphocytes lack significant AEP activity and that cleavage by AEP is dispensable for proteolytic processing of MBP in this type of cell. Instead, cathepsin (Cat) G, a serine protease that is not endogenously synthesized by B lymphocytes, is internalized from the plasma membrane and present in lysosomes from human B cells where it represents a major functional constituent of the proteolytic machinery. CatG initialized and dominated the destruction of intact MBP by B cell-derived lysosomal extracts, degrading the immunodominant MBP epitope and eliminating both its binding to MHC class II and a MBP-specific T cell response. Degradation of intact MBP by CatG was not restricted to a lysosomal environment, but was also performed by soluble CatG. Thus, the abundant protease CatG might participate in eliminating the immunodominant determinant of MBP. Internalization of exogenous CatG represents a novel mechanism of professional APC to acquire functionally dominant proteolytic activity that complements the panel of endogenous lysosomal enzymes.

Purpose To assess the safety and efficacy of CT-guided brachytherapy alone or in combination with laser-induced thermotherapy (LITT) in patients with liver malignancies. Methods and materials Thirty-seven patients presented with 36 liver metastases and two primary liver carcinomas. Twenty-one patients were treated with CT-guided high-dose-rate brachytherapy alone using a 192 Ir source. Sixteen patients received brachytherapy directly after MRI-guided LITT. The indications for brachytherapy alone were a tumor size >5 cm, adjacent central bile duct or adjacent major vessels causing unfavorable cooling effects for thermal ablation, and technical failures of LITT. The dosimetry for brachytherapy was performed using three-dimensional CT data acquired after percutaneous applicator positioning. On average, a minimal dose of 17 Gy inside the tumor margin was applied (range, 10–20 Gy). Results The mean tumor size was 4.6 cm (range, 2.5–11 cm). The mean liver volume receiving ≥5 Gy was 16% (range, 2–40%) of the total liver. Severe complications were recorded in 2 patients (5%). One patient developed acute liver failure possibly related to accidental continuation of oral capecitabine treatment. Another patient demonstrated obstructive jaundice owing to tumor edema after irradiation of a metastasis adjacent to the bile duct bifurcation. A commonly encountered moderate increase of liver enzymes was greatest in patients with combined treatment. The local control rate after 6 months was 73% and 87% for combined treatment and brachytherapy alone, respectively. Conclusion CT-guided brachytherapy using three-dimensional CT data for dosimetry is safe and effective alone or in combination with LITT. Brachytherapy as a stand-alone treatment displayed genuine advantages over thermal tumor ablation.

Using Zn2+ ions as new method, several FUs have been isolated from molluscan Hc Rapana venosa without formation of non-functional proteolytic side products. N-terminal sequences of these fragments in comparison with FUS from other gastropodan Hcs show a very high degree of structural identity. Four Fus, purified from enzyme-treated structural subunits RvH1 and RvH2 (RvH1-a, RvH1-f, RvH2-a and RvH2-e) show identical N-terminal sequences compared to fragments isolated after treatment with Zn2+ ions. However, in some cases trypsin cleaves RvH chains at different positions if compared to the Zn2+ treatment. To analyze the oligosaccharide composition of two FUS from the first structural subunit of Rapana Hc, RvH1-a and RvH1-f, several techniques were applied: capillary electrophoresis, MALDI-MS, ESI-MS in combination with glycosidase digestions. On basis of these results and the determined amino acid sequence two N-linkage sites were identified in the FU RvH1-a, but only one in the FU RvH1-f.

Unter dem Motto „Bewegung ist Leben“ fand im September die 8. Summer School der Deutschen Gesellschaft fur Orthopadie und Unfallchirurgie (DGOU) und des Berufsverbands fur Orthopadie und Unfallchirurgie (BVOU) in Frankfurt am Main statt. Prof. Dr. Andrea Meurer, Prof. Dr. Ingo Marzi und Prof. Dr. Alexander Beck haben die Summer School geleitet, durchgefuhrt wurde sie in Kooperation mit dem Jungen Forum O & U.

The lysine in position 66 of the β chain of hemoglobin (Hb) is located within the heme-binding pocket and is functionally important for oxygen binding (1). The lysine is exceptionally prone to glycation (2) and to modification by advanced glycation endproducts. In this journal, Iwamoto et al. (3) recently proposed an automated immunologic assay for Hb that is specifically advanced glycation endproduct-modified at Lys66. Here we report on a novel Hb variant at this position [β66(E10) Lys→Asn] found in a 73-year-old Caucasian patient with a 9-month history of type 1 diabetes mellitus secondary to recurrent necrotizing pancreatitis and choledocholithiasis. He was referred to us because of insufficient metabolic control: in the previous 4-month period, his fasting blood glucose (FBG) had been in the range of 6.3–13.6 mmol/L and his postprandial blood glucose had been in the range of 8.8–17.0 mmol/L. On admission, his FBG was 13.6 mmol/L. The blood smear results, complete blood count, reticulocyte count, and other markers for chronic anemia or hemolysis were within the appropriate reference intervals. HbF (0.6%) and HbA2 (2.1%) were not increased. The initial measurement of HbA1c by cation-exchange HPLC (Tosoh HLC723-GHb A1c Version 2.2; Tosoh/Eurogenetics) gave a markedly low HbA1c value of 6.0% compared with 9.0% measured by the Bayer DCA immunologic method (Bayer Diagnostics). The chromatogram (Fig. 1A⇓ ) obtained with the routinely used 3-min HPLC program showed an abnormal peak interfering with the HbA1c fraction but no other major peak suspicious for a heterozygous Hb variant. Under optimized insulin treatment, the patient remained moderately hyperglycemic. Despite the patient’s persistent mild hyperglycemia, in subsequent HbA1c determinations under 3 and 6 months of optimized insulin treatment, the HPLC method consistently …

In the present study a rapid, reproducible and robust capillary electrophoresis (CE) procedure for the quantification of apolipoprotein A-I (Apo A-I) in serum without pretreatment has been developed (total run time, 11 min). The coefficients of variation (CV; n = 10) for the relative peak area are 1.8% at a concentration of 145 mg/dL and 1.6% at 196 mg/dL; and for the inter-assay 8.9% at 161 mg/dL (10 consecutive days), i.e., similar to the CVs of a high-throughput immunonephelometric routine assay. The CV for the migration time is 0.4% (n = 20). The robustness of the CE approach was tested in patient samples with hemolysis, hyperbilirubinemia and hyperlipidemia. A comparison of 99 Apo A-I serum values with results of a fixed-time immunonephelometric routine assay showed a positive constant bias of 60% (mean) for the immunonephelometric values, no deviation from linearity, but significant deviations in several samples. Investigations on interferences in the CE analyses gave no evidence that CE failed. Our study shows that CE is amenable to a fast analysis and a reproducible and reliable quantification of Apo A-I level in sera of various clinical samples.

Recently, we reported a fast on-line alkaline micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric approach for sensitive phosphopeptide screening of a tryptic digested protein and subsequent characterization of the identified phosphopeptide. Based on this study, we now applied an improved method for the identification of phosphorylation sites in insulin receptor substrate 1, an important mediator in insulin signal transduction which was phosphorylated in vitro by protein kinase C-ζ. The approach consists of an on-line alkaline negative-ion micro-liquid chromatography/electrospray-atmospheric pressure ionization/collision-induced dissociation/mass spectrometric hybrid scan experiment using a triple-quadrupole mass spectrometer with fractionation and subsequent off-line nanoES-MS (ion trap) analysis of the phosphopeptide-containing fractions. During the liquid chromatography (LC)/ES-MS experiment, the phosphopeptides of the enzymatic digest mixture of the studied insulin receptor substrate 1 fragment were detected under high skimmer potential (API-CID) using phosphorylation-specific m/z 79 marker ions as well as the intact m/z-values of the peptides which were recorded under low skimmer potential. Subsequently, the targeted fractions were analyzed by off-line nanoES-MS/MS and MS3. Using this approach, serine 318 was clearly identified as a major in vitro protein kinase C-ζ phosphorylation site in the insulin receptor substrate −1 fragment. Together, our results indicate that the applied strategy is useful for unequivocal and fast analysis of phosphorylation sites in low abundant signaling proteins.

The synthesis of the new chiral ligand (3,5-di-tertbutylpyrazol-1-yl)(3 ,5 -dimethylpyrazol-1-yl)acetic acid (bpaHtBu2,Me2) (4) has been achieved. Two different synthetic routes to its precursor 3,5-di-tert-butyl-1-[(3,5-dimethyl-1Hpyrazol-1-yl)methyl]-1H-pyrazole (bpmtBu2,Me2) (3) are reported. Deprotonation at the methylene group, followed by reaction with carbon dioxide, yielded a racemic mixture of 4. The chemical behaviour of bis(3,5-di-tert-butylpyrazol-1yl)acetic acid (bdtbpzaH) (2) and the new chiral N,N,O scorpionate ligand 4 involving their coordination to zinc ions was studied. [Zn(bpatBu2,Me2)Cl] (5) was formed from a mixture of ZnCl2, 4 and base. Reaction of bis(3,5-di-tert-butylpyrazol-1yl)acetic acid (bdtbpzaH) (2) with Zn(CH3)2 or Zn(CH2CH3)2 gave the alkylzinc complexes [Zn(bdtbpza)(CH3)] (6) and

The phosphorylation of serine-/threonine-residues of insulin receptor substrate-1 (IRS-1) by protein kinase C-ζ (PKC-ζ), which leads to an increased tyrosine dephosphorylation of IRS-1, has been recently identified as a novel hyperinsulinemia-induced negative feed-back regulation of the insulin signalling pathway. To identify the underlying molecular mechanism, we investigated the serine-/threonine-residues in IRS-1 phosphorylated by PKC-ζ. Because the activity of the tyrosine phosphatase SHP-2, which dephosphorylates IRS-1, is regulated by the binding to the C-terminal part of IRS-1 (IRS-1c) this region was studied. In the in vitro assay the atypical PKC ζ phosphorylates IRS-1c comparable to PKC-θ and -β1, whereas PKC-β2 showed lower and PKC-i and PKC-α showed negligible phosphorylation. We observed one major 32P-motif labelled by PKC-ζ, containing four phosphorylated serine/threonine residues (Ser 1215, 1216, 1220 and Thr 1221) near the C-terminal end of IRS-1. These sites are closely adjacent to tyrosine 1222, which represents one of the two binding sites for the protein tyrosine phosphatase SHP-2. In conclusion, the PKC-ζ-dependent in vitro phosphorylation of IRS-1 at a site closely situated to a crucial binding site of the tyrosine phosphatase SHP-2 suggests the modulation of the dephosphorylation activity of SHP-2 by PKC-ζ. Furthermore, our findings can contribute to the explanation of the complex positive and negative regulatory action of PKC-ζ on insulin signalling.

A rapid on-line method for the identification of phosphorylated peptides in enzymatic protein digests by specific marker ion signals is described. In our study we investigated the use of alkaline conditions together with a previously described method for selective and sensitive detection of phosphopeptide ions combining high-performance capillary liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS).1,, 2 Phosphorylation-specific marker ions (m/z 79, PO3−, and m/z 97, H2PO4−) were generated by skimmer collision-induced dissociation (sCID) in the negative-ion mode. The method was evaluated and validated for mono-phosphorylated synthetic peptides using different alkaline pH values and CID offsets. Alkaline conditions (pH 10.5) enhance the generation of phosphopeptide-specific fragment ions from serine- and tyrosine-phosphorylated peptides, and enable the use of m/z 79 (PO3−) and m/z 97 (H2PO4−) as phosphorylation-specific marker traces. Note that HPLC separation in trifluoroacetic acid containing solvents impairs the use of m/z 97 (C2F3O− fragment ion at m/z 97) as a phosphorylation-specific marker. The optimized method was applied for the detection of phosphorylated peptides in a tryptic β-casein digest. The expected mono- and tetra-phosphorylated peptides were detected and rapidly identified by µLC/ESI-sCID-MS and µLC/ESI-MS analysis. Copyright © 2001 John Wiley & Sons, Ltd.

The chemical behavior of two bis(3,5-dialkylpyrazol-1-yl)acetic acids and their coordination properties towards ZnCl2 and FeCl2 was studied. Reaction of bis(3,5-dimethylpyrazol-1-yl)acetic acid (bdmpza) (1) with ZnCl2 and FeCl2 gave the 2:1 complexes [(bdmpza)2Zn] (4) and [(bdmpza)2Fe] (5). Crystal structures of both complexes were obtained. In contrast, the sterically more hindered ligand bis(3,5-di-tert-butylpyrazol-1-yl)acetic acid (bdtbpza) (3) coordinates only once to zinc(II) resulting in the complex [(bdtbpza)ZnCl] (6). This provides a structural model complex for the active sites of zinc enzymes such as thermolysin or carboxypeptidase. The good agreement with these active sites was demonstrated by a crystal structure of 6. To the best of our knowledge, this is the first report of zinc complexes with a monoanionic N,N,O-tridentate ligand using a carboxylate O-donor. Substitution of benzylthiolate for the chloride ligand 6 yielded [(bdtbpza)Zn(SCH2Ph)] (8) indicating that the bulky tert-butyl groups do not prevent access to the zinc atom. Reaction of bdtbpza (3) with FeCl2 led to [(bdtbpza)FeCl] (7), which might serve as a structural model complex for the active sites of mononuclear non-heme iron oxidases and oxygenases such as IPNS, DAOCS or CAS.

Serine residues of the human insulin receptor (HIR) may be phosphorylated and negatively regulate the insulin signal. We studied the impact of 16 serine residues in HIR by mutation to alanine and co-overexpression in human embryonic kidney (HEK) 293 cells together with the docking proteins insulin receptor substrate (IRS)-1, IRS-2, or (SHC) Src homologous and collagen-like. As a control, IRS-1 was also cotransfected with an HIR with a juxtamembrane deletion (HIR delta JM) and therefore not containing the domain required for interaction with IRS-1. Coexpression of HIR with IRS-1, IRS-2, and SHC strongly enhanced tyrosine phosphorylation of these proteins. A similar increase in tyrosine phosphorylation was observed in cells overexpressing IRS-1, IRS-2, or SHC together with all HIR mutants except HIR delta JM and a mutant carrying exchanges of serines 1177, 1178, and 1182 to alanine (HIR1177/78/82), although this mutant showed normal autophosphorylation. Analysis of total cell lysates with anti-phosphotyrosine antibodies showed that in addition to the overexpressed substrates, other cellular proteins displayed reduced levels of tyrosine phosphorylation in these cells. To study consequences for phosphatidylinositol 3-kinase (PI 3-kinase) activation, we established stable NIH3T3 fibroblast cell lines overexpressing wild-type HIR, HIR1177/78/82, and other HIR mutants as the control. Again, HIR1177/78/82 showed normal autophosphorylation but showed a clear decrease in tyrosine phosphorylation of endogenous IRS-1 and activation of PI 3-kinase. This decrease in kinase activity also occurred in an in vitro kinase assay towards recombinant IRS-1. Finally, we performed a separation of the phosphopeptides by high-performance liquid chromatography and could not detect any differences in the profiles of HIR and HIR1177/78/82. In conclusion, we have defined a region in HIR that is important for substrate phosphorylation but not autophosphorylation. Therefore, this mutant may provide new insights into the mechanism of kinase activation and substrate phosphorylation.

The prosclerotic transforming growth factor β1 (TGF-β1) is a key factor in the induction and maintenance of fibrosis in different organs. To assess relative changes in TGF-β1 mRNA levels, the comparative kinetic reverse transcription-polymerase chain reaction strategy was used. In this method, cellular mRNA levels of the target and a house-keeping gene are reverse transcribed, amplified by the polymerase chain reaction (PCR) and the kinetics of PCR amplification are compared. Since the current determination of the PCR products, using electrophoretic separation in polyacrylamide gel, staining and scanning of the gel, is timeconsuming (≥ 5 hours) and inaccurate, we have developed a method using capillary gel electrophoresis (CGE) in combination with laser-induced fluorescence (LIF) detection for quantification of PCR-products. Using the CGE-LIF method, a minute aliquot of the PCR reaction mixture is separated and quantified within 10 min. Comparison of the values with those obtained by polyacrylamide gel electrophoresis demonstrates the improved sensitivity (> 1000 fold) and accuracy of the proposed method. The CGE-LIF procedure offers a convenient way of automated, comparative analysis of low levels of mRNA via reverse transcription PCR in low cell numbers or small amounts of tissue samples.

Insulin resistance in skeletal muscle is found in obesity and type 2 diabetes. A mechanism for impaired insulin signaling in peripheral tissues is the inhibition of insulin action through serine phosphorylation of insulin receptor substrate (Irs) proteins that abolish the coupling of Irs proteins to the activated insulin receptor. Recently, we described serine-318 as a protein kinase C (PKC)-dependent phosphorylation site in Irs1 (Ser-318) activated by hyperinsulinemia. Here we show in various cell models that the adipose hormone leptin, a putative mediator in obesity-related insulin resistance, promotes phosphorylation of Ser-318 in Irs1 by a janus kinase 2, Irs2, and PKC-dependent pathway. Mutation of Ser-318 to alanine abrogates the inhibitory effect of leptin on insulin-induced Irs1 tyrosine phosphorylation and glucose uptake in L6 myoblasts. In C57Bl/6 mice, Ser-318 phosphorylation levels in muscle tissue were enhanced by leptin and insulin administration in lean animals while in diet-induced obesity Ser-318 phosphorylation levels were already up-regulated in the basal state, and further stimulation was diminished. In analogy, in lymphocytes of obese hyperleptinemic human subjects basal Ser-318 phosphorylation levels were increased compared to lean individuals. During a hyperinsulinemic euglycemic clamp, the increment in Ser-318 phosphorylation observed in lean individuals was absent in obese. In summary, these data suggest that phosphorylation of Ser-318 in Irs1 mediates the inhibitory signal of leptin on the insulin-signaling cascade in obese subjects.

It is well established that extensive depletion of ozone, initiated by heterogenous reactions on polar stratospheric clouds (PSCs) can occur in both the Arctic and Antarctic lower stratosphere1,2,3,4,5,6,7,8,9. Moreover, it has been shown that ozone loss rates in the Arctic region in recent years reached values comparable to those over the Antarctic8,9. But until now the accumulated ozone losses over the Arctic have been the smaller, mainly because the period of Arctic ozone loss has not—unlike over the Antarctic—persisted well into springtime8,9,10. Here we report the occurrence—during the unusually cold 1995–96 Arctic winter—of the highest recorded chemical ozone loss over the Arctic region. Two new kinds of behaviour were observed. First, ozone loss at some altitudes was observed long after the last exposure to PSCs. This continued loss appears to be due to a removal of the nitrogen species that slow down chemical ozone depletion. Second, in another altitude range ozone loss rates decreased while PSCs were still present, apparently because of an early transformation of the ozone-destroying chlorine species into less active chlorinenitrate. The balance between these two counteracting mechanisms is probably a fine one, determined by small differences in wintertime stratospheric temperatures. If the apparent cooling trend in the Arctic stratosphere11 is real, more dramatic ozone losses may occur in the future.

In 2007 EU Regulation (EC) 834/2007 introduced principles and criteria for organic food processing. These regulations have been analysed and discussed in several scientific publications and research project reports. Recently, organic food quality was described by principles, aspects and criteria. These principles from organic agriculture were verified and adapted for organic food processing. Different levels for evaluation were suggested. In another document, underlying paradigms and consumer perception of organic food were reviewed against functional food, resulting in identifying integral product identity as the underlying paradigm and a holistic quality view connected to naturalness as consumers' perception of organic food quality. In a European study, the quality concept was applied to the organic food chain, resulting in a problem, namely that clear principles and related criteria were missing to evaluate processing methods. Therefore the goal of this paper is to describe and discuss the topic of organic food processing to make it operational. A conceptual background for organic food processing is given by verifying the underlying paradigms and principles of organic farming and organic food as well as on organic processing. The proposed definition connects organic processing to related systems such as minimal, sustainable and careful, gentle processing, and describes clear principles and related criteria. Based on food examples, such as milk with different heat treatments, the concept and definitions were verified. Organic processing can be defined by clear paradigms and principles and evaluated according criteria from a multidimensional approach. Further work has to be done on developing indicators and parameters for assessment of organic food quality.

Background: Fever after acute cerebral injury is associated with unfavorable functional outcome and increased mortality, but there is controversy about the optimal antipyretic treatment. This study investigated an institutional standard operating procedure (SOP) for fever treatment in stroke patients including a sequence of pharmacologic and physical interventions. Methods: A 4-step antipyretic SOP was established for patients with acute cerebral ischemia or hemorrhage and a body temperature ≧37.5°C within the first 6 days after admission. Data on the course of body temperature, duration of fever and achievement of normothermia were recorded. Results were compared to a historic control group that underwent conventional treatment. Results: A total of 77 patients (mean age 70.4 ± 14.2 yeas) received 331 antipyretic interventions. Sequential administration of paracetamol (n = 219), metamizole (n = 71) and calf packing (n = 24) resulted in a significant drop in body temperature after 60 min in each instance. In 5 of 9 cases which were refractory to previous attempts, normothermia followed the infusion of ice-cooled saline. In more than 90% of cases treated per protocol, normothermia was achieved within 120 min. Compared to conventional treatment, fever burden was significantly lower within the first 4 days after admission (p < 0.001). Conclusion: This SOP may help to optimize antipyretic treatment for stroke patients.

In this paper, we examine the S&P 500 index log-returns on short intraday time scales with three different ARMA-GARCH models. In order to forecast market risk, we describe the innovation process with tempered stable distributions which we compare to commonly used methods in financial modeling. Value-at-risk backtests are provided where we find that models based on the tempered stable innovation assumption significantly outperform traditional models in forecasting risk on short time-scales. In addition to value-at-risk, the idiosyncratic differences in average value-at-risk are compared between the models.

Die 8. Summer School der DGOU (Deutschen Gesellschaft fur Orthopadie und Unfallchirurgie) und des BVOU (Berufsverband fur Orthopadie und Unfallchirurgie) wurde am 19. und 20. September 2016 unter der wissenschaftlichen Leitung von Frau Prof. Andrea Meurer, Herrn Prof. Dr. Ingo Marzi und Herrn Prof. Alexander Beck in Kooperation mit dem Jungen Forum O&U in Frankfurt veranstaltet.

The paper proposes a method based on different security-related factors to dynamically calculate the validity period of digital certificates. Currently validity periods are most often defined statically without scientific justification. This approach is not sufficient to objectively consider the actual need for security. Therefore the approach proposed in this paper considers relevant security criteria in order to calculate a meaningful validity period for digital certificates. This kind of security assessment can be executed periodically in order to dynamically respond to changing conditions. Especially in the context of complex systems and infrastructures that have an increased need for security, privacy and availability this issue is highly relevant.

The unsaturated hydraulic conductivity of a silty sand at different initial void ratios is measured using the instantaneous profile method. The variation of the suction and volumetric water content is recorded during the infiltration process as a function of time. Accordingly, an infiltration column was developed with a height of 600 mm and an inner diameter of 170 mm. The suction and volumetric water content were measured simultaneously every 100 mm along the column by means of small tensiometers and TDRs, respectively. Hydraulic conductivity is calculated by dividing the water flow velocity by the hydraulic gradient. The soil is reconstituted from Ruedlingen (Canton Schaffhausen, Switzerland), where landslide triggering experiments were carried out in October 2008 and March 2009. The hydraulic conductivity functions are determined and the laboratory values are compared to the in-situ measurements of hydraulic conductivity carried out in the course of the landslide triggering experiments.

Background: Prehospital cooling of acute stroke patients would be ideal when associated with minor or no side effects. Therefore, we evaluated a cooling cap for the surface of head and cervical regions in awake volunteers. Methods: 10 healthy volunteers were treated by external cooling for 190 min using a gel-based cooling device. Vital signs, rectal temperature, tympanic temperature, the extent of shivering and individual perception of frostiness and discomfort were measured. Results: All participants (median age 35 years) successfully completed the treatment and experienced only mild to moderate discomfort. No serious adverse events and no shivering were noticed. There was a significant drop in the tympanic temperature to 34.68°C (difference from baseline: 1.7°C, 95% CI: 0.61–2.7°C, p = 0.001), in the rectal temperature to 36.65°C (difference from baseline: 0.65°C, 95% CI: 0.06–1.2°C, p = 0.019) and in the heart rate (difference from baseline: 15 beats/min, 95% CI: 0.63–30 beats/min, p = 0.035). Conclusion: Treatment with the cooling device was well tolerated by all participants. The technique had measurable effects on core body temperature (rectal) and tympanic temperature (may reflect temperature at the external ear and skin rather than intracranial). It can be considered as a simple therapeutic approach to patients with suspected stroke in the prehospital setting.

We present status and results of AstroGrid-D, a joint effort of astrophysicists and computer scientists to employ grid technology for scientific applications. AstroGrid-D provides access to a network of distributed machines with a set of commands as well as software interfaces. It allows simple use of computer and storage facilities and to schedule or monitor compute tasks and data management. It is based on the Globus Toolkit middleware (GT4). Chapter 1 describes the context which led to the demand for advanced software solutions in Astrophysics, and we state the goals of the project. We then present characteristic astrophysical applications that have been implemented on AstroGrid-D in chapter 2. We describe simulations of different complexity, compute-intensive calculations running on multiple sites, and advanced applications for specific scientific purposes, such as a connection to robotic telescopes. We can show from these examples how grid execution improves e.g. the scientific workflow. Chapter 3 explains the software tools and services that we adapted or newly developed. Section 3.1 is focused on the administrative aspects of the infrastructure, to manage users and monitor activity. Section 3.2 characterises the central components of our architecture: The AstroGrid-D information service to collect and store metadata, a file management system, the data management system, and a job manager for automatic submission of compute tasks. We summarise the successfully established infrastructure in chapter 4, concluding with our future plans to establish AstroGrid-D as a platform of modern e-Astronomy., Comment: 14 pages, 12 figures Subjects: data analysis, image processing, robotic telescopes, simulations, grid. Accepted for publication in New Astronomy

Ob Qualitat messbar ist, daruber streiten sich die Gelehrten. Die DGOU will Konzepte vorschlagen, die verbindliche Regeln vorgeben, um die numerische Mindestqualitat durch Prozess- und Strukturqualitat zu ersetzen, bevor uns von ubergeordneten Stellen Kriterien auferlegt werden. Hierdurch sollen die Frage der Versorgung in der Flache, die Qualitat der erbrachten Leistung definiert und die Sicherheit der Patienten verbessert werden.

As observed in most molluscan hemocyanins, high-mannose type glycans were identified in hemocyanins from Rapana venosa (RvH), Helix lucorum (HlH) and keyhole limpet (Megatura crenulata). In addition, a glycan with a branching structure containing xylose, fucose and terminal methyl hexose was identified in β-HlH. We have examined the immuno-adjuvant properties of hemocyanins, their derivatives and conjugates associated with the cell mediated immunity in experimental tumor-bearing animals with ascites tumor of Guerin. After immunization of the animals with the experimental vaccine preparations, the highest values of splenic lymphocytes were observed in groups immunized with the conjugates RvH-TAg, β-HlH-TAg and KLH-TAg (42.3%; 40.8% and 40.58%, respectively) than with the native hemocyanins (36.5%; 35.1% and 32.4%, respectively). The immunization of rats with the hemocyanins β-HlH, RvH and KLH and their conjugates, prolonged the median survival time of tumor-bearing animals compared with non-immunized animals (39, 33, 31 and 7 days, respectively). Both hemocyanins β-HlH and RvH activate the immune system of the experimental animals and therefore could be a good alternative for KLH. For this reason they could be included into the composition of non-specific anti-tumor vaccines to enhance their effectiveness.

Making applications Grid-aware (or Grid-enabled) requires the knowledge of the API of different Grid middlewares. The complexity of these systems, both in terms of underlying technology and functionality, remains high. Moreover, Grid middlewares like Globus and UNICORE are still undergoing many changes. Grid Application Toolkit (GAT), a high-level API for accessing Grid services, provides application developers with a unified, simple and middleware-independent interface to the Grid. In this paper, we present a newly developed adaptor for GAT to access UNICORE services. We also describe how the data management client DataFinder has been Grid-enabled with GAT to access remote files and submit computational jobs to the Grid. DataFinder provides primarily an easy-to-use tool for scientific data management in distributed environments. Within the project AeroGrid, a BMBF-funded project in the German D-Grid initiative, DataFinder is being used as user interface for performing complex simulations in a UNICORE-based Grid infrastructure.

Triiodobenzoic acid (TIBA) represents the core structure of most clinically used contrast agents for computed tomography and other X-ray procedures. To construct an intracellular radiopaque contrast agent, TIBA was coupled to various different positively and negatively charged fluorescein iothiocyanate (FITC)-labelled peptides. TIBA coupled to the SV40 T Antigen nuclear localization sequence (NLS) stained 80% of human glioma cells and caused cell death. This occurred with C- or N-terminal binding of TIBA and with the correct or mutant NLS. No cell death and only small numbers of stained cells (below 3 %) were observed after incubation with NLS conjugates lacking TIBA or after incubation with TIBA-conjugates containing a negatively charged polyglutamic acid stretch. TIBA-conjugates containing the Antennapedia-derived cell-penetrating peptide penetratin were only nuclearly taken up when TIBA and FITC were coupled to lysines outside the 16-amino acid peptide sequence. The study shows that intracellular TIBA may have potential as a chemotherapeutic agent rather than a contrast agent.

Human cytomegalovirus (HCMV) infection suppresses cellular immunity and results in viral persistence. Dendritic cells (DCs) are susceptible to HCMV, and the development and immune function of HCMV-infected DCs are impaired in vitro. HCMV-derived proteins interfere with different aspects of major histocompatibility complex type II (MHC II) maturation and function in genetically engineered cellular models. This study directly analysed the effect of HCMV on the MHC II-associated antigen processing and presentation machinery in HCMV-infected human DCs in vitro. HCMV-infected DCs failed to mature newly synthesized MHC II to the final stage of SDS-stable MHC II αβ dimer/peptide complexes, in contrast to mock-infected controls. MHC II biosynthesis was delayed and reduced, whilst MHC II stability remained unchanged. MHC II surface expression was decreased in the late phase of HCMV infection. In addition, infected DCs decreased the transcription rate of the MHC II-associated proteases cathepsins S, Z, B, H and L and asparagine-specific endopeptidase (AEP). This translated into reduced protein expression of cathepsins H and S, as well as AEP, and less-efficient proteolytic degradation of a peptide substrate by endocytic proteases from HCMV-infected DCs in vitro. Thus, HCMV infection interferes with MHC II biosynthesis and maturation, as well as with the expression and function of endocytic proteases in infected DCs.

The seven N-terminal amino acids AVPIAQK (SmacN7) of the mitochondrial protein Smac (second mitochondria-derived activator of caspase) promote caspase activation by binding specifically to inhibitor of apoptosis proteins (IAPs) and blocking their inhibitory activity. SmacN7 cannot pass through the cell membrane, but to be of therapeutic use it would be essential for it to enter the cell. To achieve transmembrane transport of SmacN7 we coupled it to a novel fluorescein isothiocyanate (FITC)-labelled transmembrane transport peptide RRRRK(FITC)RRRR via ss-alanine to produce the conjugate AVPIAQKssA RRRRK(FITC)RRRR. Because IAPs are much more strongly expressed in the cytoplasm of tumor cells, we expected this conjugate to produce staining of the cytoplasm, and for this to be stronger in tumor cells than in healthy cells. Surprisingly, we found strong nuclear uptake of the Smac conjugate and of the transport peptide alone without subsequent release in both tumor cells and healthy cells from the bladder, prostate, and brain. This was accompanied by cell death. In contrast to expectations, it appears that the apoptotic effects observed do not result from the SmacN7 cargo alone.

Cellular and nuclear uptake of dual labelled conjugates could be of great value for chemotherapy and cancer diagnostics. Therefore we designed conjugates in which gadolinium (Gd)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA), a contrast agent for magnetic resonance imaging and fluorescein isothiocyanate (FITC), a fluorescence marker were coupled to membrane translocation sequences (MTS). The MTSs we employed were the third helix of the Antennapedia homeodomain, the HIV-1 Tat peptide and the N-myristoylated HIV-1 Tat peptide. We used confocal laser scanning microscopy, fluorescence activated cell sorting, magnetic resonance imaging (MRI) and viability tests to examine the cellular and nuclear uptake of these conjugates into U373 glioma cells, as well as their cytotoxic effects. We found that the Antennapedia conjugate was taken up by no more than 20% of the cells. The HIV-1 Tat conjugate showed even lower uptake into less than 3% of cells. Interestingly, N-myristoylation of the HIV-1 Tat conjugate drastically improved its cellular uptake. Up to 70% of cells showed cellular and nuclear uptake of the N-myristoylated HIV-1 Tat conjugate. Conjugate cytotoxicity appears to correlate with cellular uptake.

Vertebrate metallothioneins are found to contain Zn(II) and variable amounts of Cu(I), in vivo, and are believed to be important for d10-metal control. To date, structural information is available for the Zn(II) and Cd(II) forms, but not for the Cu(I) or mixed metal forms. Cu(I) binding to metallothionein-1 has been investigated by circular dichroism, luminescence and 1H NMR using two synthetic fragments representing the alpha- and the beta-domain. The 1H NMR data and thus the structures of Zn4alpha metallothionein (MT)-1 and Zn3betaMT-1 were essentially the same as those already published for the corresponding domains of native Cd7MT-1. Cu(I) titration of the Zn(II)-reconstituted domains provided clear evidence of stable polypeptide folds of the three Cu(I)-containing alpha- and the four Cu(I)-containing beta-domains. The solution structures of these two species are grossly different from the structures of the starting Zn(II) complexes. Further addition of Cu(I) to the two single domains led to the loss of defined domain structures. Upon mixing of the separately prepared aqueous three and four Cu(I) loaded alpha- and beta-domains, no interaction was seen between the two species. There was neither any indication for a net transfer of Cu(I) between the two domains nor for the formation of one large single Cu(I) cluster involving both domains.