5-Hydroxyindoleacetic acid and homovanillic acid concentrations in cerebrospinal fluid in patients with Alzheimer's disease, depression and mild cognitive impairment

In this study we investigated the cerebrospinal fluid (CSF) concentrations of 5-hydroxyindoleacetic acid (5-HIAA) and homovanillic acid (HVA) in Alzheimer (AD) patients (n=75), patients with mild cognitive impairment (MCI, n=9) and patients with depression (n=7). CSF HVA was significantly elevated in AD with depression (Geriatric Depression Scale, 15 point version GDS>5) in comparison to AD without depression (p<0.05, ANOVA) and CSF HVA showed a significant positive correlation with the GDS score of AD-patients (p=0.03, Spearman Rho: 0.38, Spearman Rank Correlation). In the group of AD patients CSF 5-HIAA was positively correlated with cerebrospinal fluid beta-amyloid 1-42 (Abeta42), p<0.05, Spearman Rho: 0.3, Spearman Rank Correlation, but not with CSF tau. Additionally, there was a significant positive correlation between cerebrospinal fluid 5-HIAA and HVA in the group of AD patients (p<0.0001, Rho: 0.47, Spearman Rank correlation). Neither 5-HIAA nor HVA in CSF could differentiate between mild cognitive impairment, depression and AD. The results of this study support the hypothesis that the serotonergic system plays a role in the course of AD. They further suggest an important role of dopamine metabolism in depression within AD patients.     

Differential RNA interference: replacement of endogenous with recombinant low density lipoprotein receptor-related protein (LRP)

The interpretation of experiments involving the overexpression of a recombinant cDNA is often hampered by the interference of mRNA expression from the endogenous gene locus. Unless cell lines from naturally occurring mutations or knockout mice are available, difficult and time-consuming gene targeting techniques are required to inhibit endogenous gene expression. Using a method we refer to as "differential RNA interference" we demonstrate that RNA interference can be used to selectively suppress endogenous gene expression without affecting the expression of a co-transfected recombinant version of the same protein. Functional analyses of recombinant low density lipoprotein receptor-related protein (LRP) to study its involvement in lipid metabolism have been shown to be extremely difficult due to its large cDNA and the unavailability of suitable LRP-deficient cell lines. We constructed an expression vector containing the full-length coding sequence of human LRP fused to EGFP and a vector expressing small hairpin RNA directed against the 3'-untranslated region of the wild-type human LRP mRNA (LRP-shRNA). When overexpressed, EGFP-tagged LRP colocalizes with endogenous LRP and stimulates the uptake of LRP ligands. Overexpression of LRP-shRNA vectors significantly inhibits LRP expression, as judged by quantitative RT-PCR, Western blot and immunofluorescence analysis, and it dramatically decreases receptor-associated protein (RAP) uptake. Finally, co-transfection of EGFP-LRP and LRP-shRNA vectors demonstrates selective inhibition of endogenous LRP expression without affecting simultaneous expression of recombinant LRP protein. Thus, utilization of "differential RNA interference" provides a new experimental approach to selectively study the function of any recombinant protein in any given cell line without interference of endogenous protein expression.     

Sequential roles of Cdc42, Par-6, aPKC, and Lgl in the establishment of epithelial polarity during Drosophila embryogenesis

How epithelial cells subdivide their plasma membrane into an apical and a basolateral domain is largely unclear. In Drosophila embryos, epithelial cells are generated from a syncytium during cellularization. We show here that polarity is established shortly after cellularization when Par-6 and the atypical protein kinase C concentrate on the apical side of the newly formed cells. Apical localization of Par-6 requires its interaction with activated Cdc42 and dominant-active or dominant-negative Cdc42 disrupt epithelial polarity, suggesting that activation of this GTPase is crucial for the establishment of epithelial polarity. Maintenance of Par-6 localization requires the cytoskeletal protein Lgl. Genetic and biochemical experiments suggest that phosphorylation by aPKC inactivates Lgl on the apical side. On the basolateral side, Lgl is active and excludes Par-6 from the cell cortex, suggesting that complementary cortical domains are maintained by mutual inhibition of aPKC and Lgl on opposite sides of an epithelial cell.     

Laser light-scattering evidence for an altered association of beta B1-crystallin deamidated in the connecting peptide

Deamidation is a prevalent modification of crystallin proteins in the vertebrate lens. The effect of specific sites of deamidation on crystallin stability in vivo is not known. Using mass spectrometry, a previously unreported deamidation in beta B1-crystallin was identified at Gln146. Another deamidation was investigated at Asn157. It was determined that whole soluble beta B1 contained 13%-17% deamidation at Gln146 and Asn157. Static and quasi-elastic laser light scattering, circular dichroism, and heat aggregation studies were used to explore the structure and associative properties of recombinantly expressed wild-type (wt) beta B1 and the deamidated beta B1 mutants, Q146E and N157D. Dimer formation occurred for wt beta B1, Q146E, and N157D in a concentration-dependent manner, but only Q146E showed formation of higher ordered oligomers at the concentrations studied. Deamidation at Gln146, but not Asn157, led to an increased tendency of beta B1 to aggregate upon heating. We conclude that deamidation creates unique effects depending upon where the deamidation is introduced in the crystallin structure.     

Interleukin-18 enhances the production of interferon-gamma (IFN-gamma) by allergen-specific and unspecific stimulated cord blood mononuclear cells

BACKGROUND: IL-18 is a pleiotropic cytokine involved in the polarisation of T-cell response. This study was performed to determine whether or not IL-18 is detectable in phytohemagglutinin (PHA) or betalactoglobulin (BLG) stimulated supernatants of cord blood mononuclear cells (CBMC) and to study the in vitro effect of IL-18 on the interferon (IFN)-gaamma and IL-13 release of CBMC of healthy neonates. METHODS: CBMC of neonates were isolated by Ficoll density centrifugation. The cytokines IFN-gamma, IL-13 and IL-18 in the cell culture supernatants were measured using the ELISA technique following stimulation with a unspecific (PHA 20 microg/ml) and an allergen-specific stimulus (BLG 25 microg/ml). In order to study the in vitro effect of IL-18, CBMC were stimulated either with medium alone or with IL-18, IL-18 + PHA and IL-18 + BLG. RESULTS: IL-18 levels in supernatants of CBMC were low and did not vary significantly between unstimulated and PHA or BLG stimulated cell cultures (median 21.4; 23.5 and 15.5 pg/ml, respectively). IFN-gamma and IL-13 levels were significantly higher in response to PHA and BLG (PHA: IFN-gamma, 6154; IL-13, 4357; BLG: IFN-gamma, 801; IL-13, 249 pg/ml) compared to unstimulated cell cultures. The addition of IL-18 to PHA or BLG stimulated CBMC significantly enhanced the IFN-gamma release (PHA: 6154; PHA + IL-18: 13474, p = 0.0001; BLG: 801; BLG + IL-18: 1077, p = 0.008). In comparison to incubation without IL-18, the release of IL-13 was invariable or even reduced, when CBMC were stimulated with PHA + IL-18 (4026, p = 0.16) or BLG + IL-18 (124, p = 0.0001) compared to stimulation of CBMC with PHA (4357 pg/ml) or BLG (249 pg/ml) alone. CONCLUSIONS: IL-18 is detectable in supernatants of CBMC. We observed a significant effect of IL-18 + PHA as well as IL-18 + BLG on IFN-gamma release in vitro. Based on our findings we conclude that IL-18 could act as a strong TH1-inducing factor on stimulated CBMC also in vivo.     

Metabolism of the unnatural anticancer lipid safingol,L-threo-dihydrosphingosine,in cultured cells

We studied the metabolism of radioactively labeled safingol (l-threo-dihydrosphingosine) in primary cultured neurons, B104 neuroblastoma cells, and Swiss 3T3 fibroblasts, and compared it to that of its natural stereoisomer d-erythro-dihydrosphingosine. Both sphingoid bases are used as biosynthetic precursors for complex sphingolipids, albeit to different rates. Whereas a considerable amount of the natural sphingoid base is also directed to the catabolic pathway (20-66%, cell type dependent), only a minor amount of the nonnatural safingol is subjected to catabolic cleavage, most of it being N-acylated to the respective stereochemical variant of dihydroceramide. Interestingly, N-acylation of safingol to l-threo-dihydroceramide is less sensitive to fumonisin B1 than the formation of the natural d-erythro-dihydroceramide. In addition, safingol-derived l-threo-dihydroceramide, unlike its physiologic counterpart, is not desaturated. Most of it either accumulates in the cells (up to 50%) or is used as a biosynthetic precursor of the respective dihydrosphingomyelin (up to 45%). About 5% is, however, glucosylated and channeled into the glycosphingolipid biosynthetic pathway. Our results demonstrate that, despite its nonnatural stereochemistry, safingol is recognized and metabolized preferentially by enzymes of the sphingolipid biosynthetic pathway. Furthermore, our data suggest that the cytotoxic potential of safingol is reduced rather than enhanced via its metabolic conversion.     

A long-term porcine model for measurement of gastrointestinal motility

Animal models have become an essential tool in the investigations of gut motility under experimental conditions. To determine the influence of various anaesthetic drugs on the motility pattern of the gastroduodenal tract, a new long-term model has had to be developed for allowing measurements in conscious and unrestrained as well as in sedated and analgosedated pigs. Since mechanical ventilation influences gut motility, it was necessary that this animal model enabled the investigation of the effect of drugs causing sedation and analgosedation during spontaneous breathing. Seven male, castrated pigs, German landrace, 32-40 kg bodyweight (BW) were investigated in this study. After habituation of the pigs to local housing conditions over 5 days, the animals were trained over 4 days to prepare for experimental situations and investigators. Pigs were inserted with a central venous catheter and with percutaneous enterogastrostomy (PEG) under general anaesthesia. Intestinal motility was measured by intraluminal impedancometry. The catheter was introduced over the PEG into the stomach and positioned into the duodenum by duodenoscopy. Measurements were done in conscious, unrestrained pigs and with sedated, and analgosedated animals on subsequent days. The habituation and training of the pigs to the investigators and for the laboratory conditions took between 7 and 9 days. The initial anaesthesia protocol for the instrumentation using remifentanil/propofol led to pyloric spasm and was thus unsuitable for duodenal intubation with an endoscope. In contrast, a combination of ketamine/propofol enabled this procedure. It was practicable to measure gut motility in conscious, unrestrained pigs. Spontaneous breathing was sufficient under propofol sedation and analgosedation using fentanyl-propofol. Systematically local application of polividon iodine in the area of the subcutaneous catheters avoided the necessity of using systemic prophylactic antibiotics. In conclusion, the habituation and training for 9 days enabled the measurement of gut motility by intraluminal impedancometry in conscious pigs. The insertion of the catheter was done during general anaesthesia using a combination of propofol and ketamine. For the future determination of gut motility performed under general anaesthesia, each sedation and analgosedation concept has to be evaluated to see whether it allows spontaneous breathing or whether mechanical ventilation is necessary.     

New cationic lipids form channel-like pores in phospholipid bilayers

Two representatives of a new class of cationic lipids were found to have high pore-forming activity in planar bilayer membranes. These molecules, called BHHD-TADC and BHTD-TADC, have qualitatively similar effects on phospholipid membranes. Addition of 2.5-5 micro M of either of them to the membrane bathing solutions resulted in formation of long-lived anion-selective pores with conductance in the range 0.1-2 nS in 0.1 M KCl. Pore formation was found to be dependent on the potential applied to the membrane. When negative potential was applied to membrane at the side of addition, the rate of pore formation was much lower compared to when the positive potential was applied. Dependence of pore formation on compound concentration was highly nonlinear, indicating that this process requires assembly of molecules in the membrane. Addition of any of these compounds on both sides of the membrane increased the efficiency of pore formation by one to two orders of magnitude. Pore formation was strongly pH dependent. Although pores were formed with high efficiency at pH 6.5, only occasional fluctuations of membrane conductance were observed at pH 7.5. Possible mechanisms of new compounds biological activity are discussed.     

Ribosomal crystallography: from poorly diffracting microcrystals to high-resolution structures

The cellular organelles translating the genetic code into proteins, the ribosomes, are large, asymmetric, flexible, and unstable ribonucleoprotein assemblies, hence they are difficult to crystallize. Despite two decades of intensive effort and thorough searches for suitable sources, so far only three crystal types have yielded high-resolution structures: two large subunits (from an archaean and from a mesophilic eubacterium) and one thermophilic small subunit. These structures have added to our understanding of decoding, have revealed dynamic aspects of the biosynthetic process, and have indicated the strategies adopted by ribosomes for interacting between themselves as well as with inhibitors, factors and substrates.