Verification and characterization of an alternative low density lipoprotein receptor-related protein 1 splice variant.

BACKGROUND: Low density lipoprotein (LDL) receptor-related protein 1 (LRP1) is a ubiquitously expressed multi-ligand endocytosis receptor implicated in a wide range of signalling, among others in tumour biology. Tumour-associated genomic mutations of the LRP1 gene are described, but nothing is known about cancer-associated expression of LRP1 splice variants Therefore, the focus of this study was on an annotated truncated LRP1 splice variant (BC072015.1; NCBI GenBank), referred to as smLRP1, which was initially identified in prostate and lung carcinoma. METHODS: Using PCR and quantitative PCR, the expression of LRP1 and smLRP1 in different human tissues and tumour cell lines was screened and compared on tumour biopsies of head and neck squamous cell carcinoma (HNSCC). Using a recently developed anti-smLRP1 antibody, the expression of the putative LRP1 protein isoform in tumour cell lines in Western blot and immunofluorescence staining was further investigated. RESULTS: The alternative transcript smLRP1 is ubiquitously expressed in 12 human cell lines of different origin and 22 tissues which is similar to LRP1. A shift in expression of smLRP1 relative to LRP1 towards smLRP1 was observed in most tumour cell lines compared to healthy tissue. The expression of LRP1 as well as smLRP1 is decreased in HNSCC cell lines in comparison to healthy mucosa. In vitro results were checked using primary HNSCC. Furthermore, the expression of the protein isoform smLRP1 (32 kDa) was confirmed in human tumour cell lines. CONCLUSIONS: Similar to LRP1, the truncated splice variant smLRP1 is ubiquitously expressed in healthy human tissues, but altered in tumours pointing to a potential role of smLRP1 in cancer. Comparative results suggest a shift in expression in favour of smLRP1 in tumour cells that warrant further evaluation. The protein isoform is suggested to be secreted.

A web-based e-learning programme for training external post-mortem examination in curricular medical education.

In Germany, the external post-mortem examination is considered a medical duty and may be performed by any licensed physician. Concerning legal medicine as a curricular subject in teaching medical students, the external post-mortem examination is regarded a core area. At the University of Müenster, 15 virtual cases of death have been developed by using the web-based Inmedea Simulator. The programme allows performing all relevant steps in executing a complete external post-mortem examination. A particular importance was attached to the aspect of training users in approaching the subject in a systematic way to interpret significant forensic findings correctly and to comprehend their medico-legal implications. The programme was used for the first time in the academic term of 2010/2011. The overall reception of the programme by the medical students resulted to be positive in a first evaluation.

SnAvi--a new tandem tag for high-affinity protein-complex purification.

Systematic tandem-affinity-purification (TAP) of protein complexes was tremendously successful in yeast and has changed the general concept of how we understand protein function in eukaryotic cells. The transfer of this method to other model organisms has been difficult and may require specific adaptations. We were especially interested to establish a cell-type-specific TAP system for Caenorhabditis elegans, a model animal well suited to high-throughput analysis, proteomics and systems biology. By combining the high-affinity interaction between in vivo biotinylated target-proteins and streptavidin with the usage of a newly identified epitope of the publicly shared SB1 monoclonal antibody we created a novel in vivo fluorescent tag, the SnAvi-Tag. We show the versatile application of the SnAvi-Tag in Escherichia coli, vertebrate cells and in C. elegans for tandem affinity purification of protein complexes, western blotting and also for the in vivo sub-cellular localization of labelled proteins.

Curcumin inhibits glyoxalase 1: a possible link to its anti-inflammatory and anti-tumor activity.

BACKGROUND: Glyoxalases (Glo1 and Glo2) are involved in the glycolytic pathway by detoxifying the reactive methylglyoxal (MGO) into D-lactate in a two-step reaction using glutathione (GSH) as cofactor. Inhibitors of glyoxalases are considered as anti-inflammatory and anti-carcinogenic agents. The recent finding that various polyphenols modulate Glo1 activity has prompted us to assess curcumin's potency as an Glo1 inhibitor. METHODOLOGY/PRINCIPAL FINDINGS: Cultures of whole blood cells and tumor cell lines (PC-3, JIM-1, MDA-MD 231 and 1321N1) were set up to investigate the effect of selected polyphenols, including curcumin, on the LPS-induced cytokine production (cytometric bead-based array), cell proliferation (WST-1 assay), cytosolic Glo1 and Glo2 enzymatic activity, apoptosis/necrosis (annexin V-FITC/propidium iodide staining; flow cytometric analysis) as well as GSH and ATP content. Results of enzyme kinetics revealed that curcumin, compared to the polyphenols quercetin, myricetin, kaempferol, luteolin and rutin, elicited a stronger competitive inhibitory effect on Glo1 (K(i) = 5.1+/-1.4 microM). Applying a whole blood assay, IC(50) values of pro-inflammatory cytokine release (TNF-alpha, IL-6, IL-8, IL-1beta) were found to be positively correlated with the K(i)-values of the aforementioned polyphenols. Moreover, whereas curcumin was found to hamper the growth of breast cancer (JIMT-1, MDA-MB-231), prostate cancer PC-3 and brain astrocytoma 1321N1 cells, no effect on growth or vitality of human primary hepatocytes was elucidated. Curcumin decreased D-lactate release by tumor cells, another clue for inhibition of intracellular Glo1. CONCLUSIONS/SIGNIFICANCE: The results described herein provide new insights into curcumin's biological activities as they indicate that inhibition of Glo1 by curcumin may result in non-tolerable levels of MGO and GSH, which, in turn, modulate various metabolic cellular pathways including depletion of cellular ATP and GSH content. This may account for curcumin's potency as an anti-inflammatory and anti-tumor agent. The findings support the use of curcumin as a potential therapeutic agent.

Oligomeric procyanidins of French maritime pine bark extract (Pycnogenol) effectively inhibit alpha-glucosidase.

The standardized maritime pine bark extract (Pycnogenol) was reported to exert clinical anti-diabetic effects after peroral intake. However, an increased insulin secretion was not observed after administration of the extract to patients. Our aim was to elucidate whether the described clinical effects of Pycnogenol are related to inhibition of alpha-glucosidase. Therefore, we analyzed the inhibitory activity of Pycnogenol, green tea extract and acarbose towards alpha-glucosidase. Furthermore, we explored different fractions of Pycnogenol containing compounds of diverse molecular masses from polyphenolic monomers, dimers and higher oligomers to uncover which components exhibited the most pronounced inhibitory activity. We found that Pycnogenol exhibited the most potent inhibition (IC(50) about 5 microg/mL) on alpha-glucosidase compared to green tea extract (IC(50) about 20 microg/mL) and acarbose (IC(50) about 1mg/mL). The inhibitory action of Pycnogenol was stronger in extract fractions containing higher procyanidin oligomers. The results obtained assign a novel, local effect to oligomeric procyanidins and contribute to the explanation of glucose-lowering effects of Pycnogenol observed in clinical trials with diabetic patients.

Polymyxin B-conjugated alpha 2-macroglobulin as an adjunctive therapy to sepsis: Modes of action and impact on lethality.

A drug targeting both the inflammatory initiators (lipopolysaccharide; LPS) and mediators [tumor necrosis factor-alpha (TNF-alpha)] should have advantage over a "single-factor targeting strategy" in sepsis prevention trials. We have prepared conjugates of polymyxin B (PMB) and the cytokine binding protein alpha2-macroglobulin (A2M). The conjugate binds TNF-alpha as well as LPS as studied by electrophoresis and phase partitioning. Compared with free PMB, the conjugate is nontoxic to cells and does not affect the viability of human monocytes. The A2M-PMB conjugate binds to the A2M receptor (CD91/low-density lipoprotein receptor-related protein 1) with affinity similar to that of the nonmodified protein. Fluorescein isothiocyanate-labeled LPS in the presence of A2M-PMB is rapidly transported into fibroblasts for degradation via receptor-mediated endocytosis. In vitro, A2M-PMB demonstrated inhibition of LPS-induced secretion of TNF-alpha from isolated monocytes as well as in the whole blood assay. The efficacy of the drug was tested in mice after induction of acute inflammation (LPS model) and after induction of a polymicrobial sepsis by cecal ligation and puncture (CLP) model. Treatment of mice with A2M-PMB up to 250 microg/g body weight was not toxic to the animal. When the drug was administered 30 min before or 30 min after the LPS challenge, a survival rate of 90 and 70%, respectively, was obtained compared with the placebo control group (5%). A2M-PMB also protected mice after induction of polymicrobial sepsis when administered 30 min before CLP. These results support our hypothesis that A2M-PMB acts as a polyvalent drug to target different host mediators as well as sepsis inducer at the same time.

Inhibition of COX-1 and COX-2 activity by plasma of human volunteers after ingestion of French maritime pine bark extract (Pycnogenol).

There is evidence from several studies that supplementation with French maritime pine bark extract (Pycnogenol) improves inflammatory symptoms in vivo. However, the molecular pharmacological basis for the observed effects has not been fully uncovered yet. Direct inhibitory effects of plant extracts or components upon cyclooxygenase (COX) activity have been repeatedly reported, but the question remained whether sufficiently high in vivo concentrations of bioactive compounds could be achieved in humans. The purpose of the present study was to determine a possible inhibition of the enzymatic activity of COX-1 and COX-2 by serum samples of human volunteers after intake of French maritime pine bark extract. This methodology considered that the serum samples would contain any bioavailable active principle. Therefore, we obtained blood samples before and after 5 days administration of 200 mg Pycnogenol to five healthy humans. The plasma moderately inhibited both COX-1 and COX-2 activities ex vivo. In a second approach, 10 volunteers received a single dose of 300 mg Pycnogenol. Only 30 min after ingestion of the pine bark extract the serum samples induced a statistically significant increase in the inhibition of both COX-1 (P < 0.02) and COX-2 (P < 0.002). This suggests a strikingly rapid bioavailability of bioeffective compounds after oral intake of the extract. Thus, we provide evidence that Pycnogenol exerts effects by inhibition of eicosanoid generating enzymes which is consistent with reported clinical anti-inflammatory and platelet inhibitory effects in vivo. The next challenge is to identify the active principle(s) that are rapidly bioavailable in human plasma.

Characterization of novel monoclonal antibodies for prostate-specific antigen (PSA) with potency to recognize PSA bound to alpha 2-macroglobulin.

BACKGROUND: Different molecular forms of prostate-specific antigen (PSA) have been used to differentiate between benign prostatic hyperplasia and prostate cancer. Detecting PSA bound to endogenous inhibitors such as alpha(1)-antichymotrypsin (ACT) and alpha(2)-macroglobulin (alpha(2)M) is often difficult because of epitope masking or sensitivity problems. Here we report the characterization of four novel mouse monoclonal antibodies (mabs) obtained by immunization with PSA-alpha(2)M complexes. Their ability to detect free PSA and PSA-inhibitor complexes was shown, and their epitopes were analyzed by phage display technology. METHODS: The properties of the mabs were studied by competition and sandwich assays and by Western blotting. Epitope mapping was performed by screening of a phage display peptide library. RESULTS: All four mabs recognized free PSA, PSA-ACT, and PSA-alpha(2)M complexes, but to various degrees. With different combinations of mabs in competition experiments, antibodies were identified that enhance binding of other mabs to PSA, forming the molecular basis of a very sensitive assay for the detection of PSA and PSA-ACT complexes. Mabs with highest reactivity for PSA-alpha(2)M were selected to establish an immunoassay for that complex. Western blot analysis revealed that all mabs recognized conformational epitopes of PSA. These findings were supported by phage display results demonstrating mimotopes in the PSA molecule. CONCLUSION: The results presented here could aid in the further development of clinically relevant assays for PSA and PSA-alpha(2)M complexes.

Antioxidant activity and inhibition of matrix metalloproteinases by metabolites of maritime pine bark extract (pycnogenol).

The procyanidin-rich maritime pine bark extract Pycnogenol has well-documented antioxidant and anti-inflammatory activity. After oral administration of Pycnogenol two major metabolites are formed in vivo, delta-(3,4-dihydroxyphenyl)-gamma-valerolactone (M1) and delta-(3-methoxy-4-hydroxyphenyl)-gamma-valerolactone (M2). We elucidated the effects of these metabolites on matrix metalloproteinases (MMPs) and determined their antioxidant activity to understand their contribution to the effects of maritime pine bark extract. We discovered strong inhibitory effects of M1 and M2 toward the activity of MMP-1, MMP-2, and MMP-9. On a microgram-per-milliliter basis both metabolites appeared more active than Pycnogenol. The metabolites were more effective than their metabolic precursor (+)-catechin in MMP inhibition. On a cellular level, we detected highly potent prevention of MMP-9 release by both metabolites, with concentrations of 0.5 microM resulting in about 50% inhibition of MMP-9 secretion. M1 was significantly more effective in superoxide scavenging than (+)-catechin, ascorbic acid, and trolox, while M2 displayed no scavenging activity. Both metabolites exhibited antioxidant activities in a redox-linked colorimetric assay, with M1 being significantly more potent than all other compounds tested. Thus, our data contribute to the comprehension of Pycnogenol effects and provide a rational basis for its use in prophylaxis and therapy of disorders related to imbalanced or excessive MMP activity.