Hartmut Kratzin

Endophilin I mediates synaptic vesicle formation by transfer of arachidonate to lysophosphatidic acid

Endophilin I is a presynaptic protein of unknown function that binds to dynamin, a GTPase that is implicated in endocytosis and recycling of synaptic vesicles. Here we show that endophilin I is essential for the formation of synaptic-like microvesicles (SLMVs) from the plasma membrane. Endophilin I exhibits lysophosphatidic acid acyl transferase (LPAAT) activity, and endophilin-I-mediated SLMV formation requires the transfer of the unsaturated fatty acid arachidonate to lysophosphatidic acid, converting it to phosphatidic acid. A deletion mutant lacking the SH3 domain through which endophilin I interacts with dynamin still exhibits LPAAT activity but no longer mediates SLMV formation. These results indicate that endophilin I may induce negative membrane curvature by converting an inverted-cone-shaped lipid to a cone-shaped lipid in the cytoplasmic leaflet of the bilayer. We propose that, through this action, endophilin I works with dynamin to mediate synaptic vesicle invagination from the plasma membrane and fission.

Serum protein profiling by SELDI mass spectrometry: detection of multiple variants of serum amyloid alpha in renal cancer patients

The molecular analysis of serum is an important field for the definition of potential diagnostic markers or disease-related protein alterations. Novel proteomic technologies such as the mass spectrometric-based surface-enhanced laser desorption/ionization (SELDI) ProteinChip® technique facilitate a rapid and reproducible analysis of such protein mixtures and affords the researcher a new dimension in the search for biomarkers of disease. Here, we have applied this technology to the study of a cohort of serum samples from well-characterized renal cell carcinoma patients for the identification of such proteins by comparison to healthy controls. We detected and characterized haptoglobin 1 α and serum amyloid α-1 (SAA-1) as disease related, in addition to an as-yet-unidentified marker of 10.84 kDa. Of particular note is the detection of multiple variants of SAA-1 in multiplex that have not been described in the sera of cancer patients. SAA-1 is detected as full-length protein, des-Arginine and des-Arginine/des-Serine variants at the N terminus by SELDI. In addition, we could also detect a low-abundant variant minus the first five N-terminal amino acids. Such variants may impact the function of the protein. We conclude the technique to be a reproducible, fast and simple mode for the discovery and analysis of marker proteins of disease in serum.

Cerebrospinal fluid‐optimized two‐dimensional difference gel electrophoresis (2‐D DIGE) facilitates the differential diagnosis of Creutzfeldt–Jakob disease

So far only the detection of 14‐3‐3 proteins in cerebrospinal fluid (CSF) is included in the diagnostic criteria for sporadic Creutzfeldt–Jakob disease (sCJD). However, this assay cannot be used for screening because of the high rate of false positive results in sCJD, and often negative results in variant CJD. To facilitate the differential diagnosis of CJD, we applied 2‐D differential gel‐electrophoresis (2‐D DIGE) as a quantitative proteomic screening system for CSF proteins. We compared 36 patients suffering from sCJD with 30 patients suffering from other neurodegenerative diseases. Sample preparation was optimized in consideration of the fact that CSF is composed of blood‐ and brain‐derived proteins, and an improved 2‐D DIGE protocol was established. Using this method in combination with protein identification by MALDI‐TOF‐MS, several known surrogate markers of sCJD like 14‐3‐3 protein, neuron‐specific enolase, and lactate dehydrogenase were readily identified. Moreover, a not yet identified protein with an approximate molecular mass of 85 kDa was found as marker for sCJD with high diagnostic specificity and sensitivity. We conclude that our proteomic approach is useful to differentiate CJD from other neurodegenerative diseases and expect that CSF‐optimized 2‐D DIGE will find broad application in the search for other brain derived proteins in CSF.

Properties of a 19-kDa Zn2+-binding protein and sequence of the Zn2+-binding domains

Ribonuclease T1 was studied by two-dimensional 1H-NMR spectroscopy. Resonance assignments were obtained for the backbone protons of 95 amino acid residues and most of its side-chain protons using sequence-specific assignment procedures. The secondary structure elements of ribonuclease T1 were identified by an investigation of medium- and long-range nuclear Overhauser effects between the backbone and C beta protons. A low-resolution three-dimensional structure of ribonuclease T1 was deduced from qualitative interpretation of long-range nuclear Overhauser effects.

Unchanged Survival Rates of 14-3-3γ Knockout Mice after Inoculation with Pathological Prion Protein

ABSTRACT The diagnosis of sporadic Creutzfeldt-Jakob disease (CJD) is based on typical clinical findings and is supported by a positive 14-3-3 Western blot of cerebrospinal fluid. However, it is not clear whether 14-3-3 indicates general neuronal damage or is of pathophysiological relevance in CJD. The fact that the 14-3-3 isoform spectrum in cerebrospinal fluid does not correspond to that found in the brain points to a regulated process. To investigate a possible role of 14-3-3 proteins in transmissible spongiform diseases, we generated a 14-3-3γ-deficient mutant mouse line by using a classical knockout strategy. The anatomy and cage behavior of the mutant mice were normal. Western blot analyses of brain homogenates revealed no changes in the protein expression of other 14-3-3 isoforms (ε, β, ζ, and η). Proteomic analyses of mouse brains by two-dimensional differential gel electrophoresis showed that several proteins, including growth hormone, 1-Cys peroxiredoxin, CCT-zeta, glucose-6-phosphate isomerase, GRP170 precursor, and α-SNAP, were differentially expressed. Mutant and wild-type mice were inoculated either intracerebrally or intraperitoneally with the Rocky Mountain Laboratory strain of scrapie, but no differences were detected in the postinoculation survival rates. These results indicate that 14-3-3γ is unlikely to play a causal role in CJD and related diseases.

Lysozyme in wine: A risk evaluation for consumers allergic to hen's egg.

Lysozyme used in wine production could present a risk for consumers allergic to hens egg. Thus, precautionary labeling of lysozyme on wines has been adopted within the European Community by updating Annex IIIa by Directive 2007/68/EC on November 27, 2007. Since no scientific data is known about the actual amounts and risks of lysozyme in wines, various in vitro efforts and skin prick tests were applied in this study to evaluate the presence of lysozyme in wines and the reactivity of those residues in allergic individuals and to fulfill the claim of updating Annex IIIa announced in Directive 2003/89/EC. Depending on the wines color (red or white wine) and fining with bentonite, which is known as an important step to remove unstable proteins mainly from white wines, diverse results were obtained concerning the amounts of lysozyme in finished wines and their in vitro and in vivo reactivity in humans allergic to hens egg.

Gas-phase sequencing after electroblotting on polyvinylidene difluoride membranes assigns correct molecular weights to myoglobin molecular weight markers

Commercially available polypeptide marker kits containing peptides generated by cyanogen bromide cleavage of either horse heart myoglobin or sperm whale myoglobin have been investigated by sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE), followed by electroblotting on polyvinylidene difluoride membranes, and gas-phase sequencing. It could be shown that the molecular weights assigned to the SDS-PAGE bands by the companies are incorrect. Arranged in descending order, the marker kits are composed of the following polypeptide fragments from myoglobin: positions 1-153, 1-131, 56-153, 56-131, 1-55, and 132-153. A polypeptide comprising residues 1-14 was not found. According to these results the log Mr versus Rf plot used for calibration must be revised. For the separation of low molecular weight polypeptides and peptides a new gel system based on the theory of multiphasic zone electrophoresis combined with a modified Coomassie staining procedure is reported.

ETA and ETB receptor antagonists synergistically increase extracellular endothelin-1 levels in primary rat astrocyte cultures

Astrocytes produce and bind endothelins (ETs), suggesting that these cells have ET autoregulatory and eliminatory functions. To further investigate these functions in primary rat astrocytes, ET-1 levels in the cell culture media (RIA/HPLC) and intracellular content of ET-1 mRNA (RT PCR) were measured under basal and stimulated (thrombin, 2.2 U/ml) conditions in the presence and absence of ETA and ETB selective antagonists (BQ123 or LU135252, and BQ788, respectively). Neither basal nor stimulated ET-1 levels in astrocyte media were influenced by ETA or ETB antagonists alone, but were significantly increased by a combination of both. ir ET-3 levels were not affected by antagonist treatment. Exogenous ET-1, added to the cultures, was rapidly cleared from the supernatant; this clearance was markedly inhibited by a combination of BQ123 and BQ788. ET-1 mRNA levels were not altered by any treatment. To conclude, in primary rat astrocyte cultures, extracellular ET-1 is cleared by binding to ET-receptors, apparently involving both, ETA and ETB sites. Thus, a blockade of the astrocytic ET eliminatory function as a consequence of the in vivo application of non-selective ET receptor antagonists may lead to increased extracellular ET levels in the brain.

Beta-amyloid peptide variants in brains and cerebrospinal fluid from amyloid precursor protein (APP) transgenic mice: comparison with human Alzheimer amyloid.

In this study, we report a detailed analysis of the different variants of amyloid-β (Aβ) peptides in the brains and the cerebrospinal fluid from APP23 transgenic mice, expressing amyloid precursor protein with the Swedish familial Alzheimer disease mutation, at different ages. Using one- and two-dimensional gel electrophoresis, immunoblotting, and mass spectrometry, we identified the Aβ peptides Aβ(1–40), -(1–42), -(1–39), -(1–38), -(1–37), -(2–40), and -(3–40) as well as minor amounts of pyroglutamate-modified Aβ (Aβ(N3pE)) and endogenous murine Aβ in brains from 24-month-old mice. Chemical modifications of the N-terminal amino group of Aβ were identified that had clearly been introduced during standard experimental procedures. To address this issue, we additionally applied amyloid extraction in ultrapure water. Clear differences between APP23 mice and Alzheimer disease (AD) brain samples were observed in terms of the relative abundance of specific variants of Aβ peptides, such as Aβ(N3pE), Aβ(1–42), and N-terminally truncated Aβ(2/3–42). These differences to human AD amyloid were also noticed in a related mouse line transgenic for human wild type amyloid precursor protein. Taken together, our findings suggest different underlying molecular mechanisms driving the amyloid deposition in transgenic mice and AD patients.

Apolipoprotein A-I as a candidate serum marker for the response to lithium treatment in bipolar disorder

The molecular basis of bipolar disorder (BD) is still unknown as is the mechanism through which lithium, the therapy of choice, exerts its effects in treatment of BD. So far, no biomarkers exist to facilitate diagnosis of BD or treatment evaluation. To investigate whether BD and its treatment with lithium leaves a characteristic signature in the serum proteome, we used SELDI‐TOF MS to analyze individual serum samples from BD patients treated with lithium (BD‐plus‐Li, n=15) or other drugs (BD‐minus‐Li, n=10) and from healthy controls (n=15). Interestingly, features of 28 kDa (one peak) and 14 kDa (three peaks) showed a decreased level in the BD‐minus‐Li group and a level restored to that of the control group in the BD‐plus‐Li group. To reveal the identity of these features, we subjected pooled serum samples from both BD groups to the 2‐D DIGE technology and identified 28 kDa apolipoprotein A‐I (apo A‐I) and three 14 kDa fragments thereof as upregulated in the BD‐plus‐Li group. Immunoturbidimetry, a routine clinical assay, verified the characteristic apo A‐I signature in individual serum samples. In conclusion, we propose apo A‐I as a candidate marker that can visualize response to lithium treatment at the serum protein level.