In vivo Toxicity Assessment of Antimicrobial Peptides (AMPs LR14) Derived from Lactobacillus plantarum Strain LR/14 in Drosophila melanogaster

Lactic acid bacteria are known to produce antimicrobial peptides (AMPs) such as bacteriocins which can be employed to control pathogens and food spoilage microorganisms. However, their possible role as toxic agents against a eukaryotic system still remains unexplored. The present study deals with the in vivo evaluation of acute toxic effect of AMPs LR14, a mixture of AMPs isolated from Lactobacillus plantarum LR/14 on Drosophila melanogaster. The fly was used as a model system to measure the extent of toxicity of these peptides. The results showed that concentrations below 10 mg/ml are not significantly effective. When exposed to 10 mg/ml of AMPs LR14, acute toxic effect and a significant delay in the developmental cycle of the fly could be observed. Also, the weight and size of the flies were significantly reduced upon ingestion of these peptides. Higher concentrations (beyond 15 mg/ml) exerted a strong larvicidal effect. Detailed analysis on larval tissues and adult germ cells of the insect revealed deformity in cellular architecture, DNA fragmentation, and premature apoptosis, confirming that the peptides have a dose-dependent toxic property. Our studies provide the first information on the role of AMPs LR14 as an insecticidal agent.     

我国肾综合征出血热疫苗生产株LR1株的全基因组序列分析

为测定我国肾综合征出血热疫苗生产株LR1株的全基因组序列 ,了解该株分子基础 ,从提取的细胞总RNA逆转录PCR扩增 ,产物纯化后克隆T载体纯化后测序 ,结果证明 ,LR1株全基因组序列由L6 5 33、M36 16、S片段的16 92个核苷酸组成 ,依各自读码框架分别编码 2 15 1、1135、42 9个氨基酸。序列同源比较分析表明 ,LR1毒株与国外HTN型毒株高度同源 ,属同一亚型 ,尤其与HTN代表株 76 - 1183个片段同源率高达 99 3%～ 99 8% ,而与国内的HTN型病毒差异较大 ,同源率仅为 79 4%～ 84 6 %。氨基酸比较也显示了同样的结果。     

Tissue-specific expression of stabilized SOLITARY-ROOT/IAA14 alters lateral root development in Arabidopsis

Auxin is important for lateral root (LR) initiation and subsequent LR primordium development. However, the roles of tissue-specific auxin signaling in these processes are poorly understood. We analyzed transgenic Arabidopsis plants expressing the stabilized mutant INDOLE-3 ACETIC ACID 14 (IAA14)/SOLITARY-ROOT (mIAA14) protein as a repressor of the auxin response factors (ARFs), under the control of tissue-specific promoters. We showed that plants expressing the mIAA14-glucocorticoid receptor (GR) fusion protein under the control of the native IAA14 promoter had the solitary-root/iaa14 mutant phenotypes, including the lack of LR formation under dexamethasone (Dex) treatment, indicating that mIAA14-GR is functional in the presence of Dex. We then demonstrated that expression of mIAA14-GR under the control of the stele-specific SHORT-ROOT promoter suppressed LR formation, and showed that mIAA14-GR expression in the protoxylem-adjacent pericycle also blocked LR formation, indicating that the normal auxin response mediated by auxin/indole-3 acetic acid (Aux/IAA) signaling in the protoxylem pericycle is necessary for LR formation. In addition, we demonstrated that expression of mIAA14-GR under either the ARF7 or the ARF19 promoter also suppressed LR formation as in the arf7 arf19 double mutants, and that IAA14 interacted with ARF7 and ARF19 in yeasts. These results strongly suggest that mIAA14-GR directly inactivates ARF7/ARF19 functions, thereby blocking LR formation. Post-embryonic expression of mIAA14-GR under the SCARECROW promoter, which is expressed in the specific cell lineage during LR primordium formation, caused disorganized LR development. This indicates that normal auxin signaling in LR primordia, which involves the unknown ARFs and Aux/IAAs, is necessary for the establishment of LR primordium organization. Thus, our data show that tissue-specific expression of a stabilized Aux/IAA protein allows analysis of tissue-specific auxin responses in LR development by inactivating ARF functions.     

Purification and partial amino acid sequence of plantaricin 1.25ααα and 1.25βββ, two bacteriocins produced by Lactobacillus plantarum TMW1.25

Two bacteriocins produced by Lactobacillus plantarum TMW1.25 have been purified by a four-step purification procedure, including ammonium sulphate precipitation and cation-exchange chromatography followed by hydrophobic-interaction chromatography on octyl sepharose. The final purification was performed by repeated reversed-phase chromatography steps which yielded two bacteriocin fractions designated plantaricin 1.25 alpha and plantaricin 1.25 beta. The molecular masses of the peptides in these fractions were 5979 and 5203 Da, respectively. Combination of the fractions did not have any synergistic effects on bacteriocin activity, indicating that they each contain a one-peptide bacteriocin. The major peptide in the alpha fraction was blocked at its N-terminus, and a partial sequence (25 residues) could only be obtained after cleavage with CNBr. This sequence did not show clear homologies with known bacteriocins. The beta peptide has been sequenced almost completely and consists, presumably, of 53 residues. This peptide displayed strong homology to the known N-terminal part of brevicin 27 produced by Lactobacillus brevis SB27. The results showed that the beta peptide contains as many as six consecutive lysine residues at the N-terminus.     

Antimicrobial peptide isolated from <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> K14 revitalizes its use in combinatorial drug therapy

The present study was performed to evaluate the antibacterial activities of an antimicrobial peptide (CSpK14) and the synergies thereof with β-lactams against vancomycin-resistant Staphylococcus aureus (VRSA) and Enterococci (VRE). Our strain was isolated from fermented food (kimchi), which is 99.79 % homologous with Bacillus amyloliquefaciens subsp. plantarum FZB42(T). CSpK14 was purified to homogeneity by diammonium sulfate precipitation, concentration, dialysis, and followed by two-stage chromatographic separation, i.e., Sepharose Cl-6B and Sephadex G-25 chromatography, and had a molar mass of ~4.6 kDa via Tricine SDS-PAGE and in situ examination. It was stable at pH 6.0–11.5 and temperature up to 80 °C. In addition, it was also stable with various metal ions, solvents, and proteases. The N-terminal amino acid sequence was H-Y-D-P-G-D-D-S-G-N-T-G and did not show any significant homology with reported peptides. However, it shows some degrees of identity with alpha-2-macroglobulin and ligand-gated channel protein from different microorganisms. CSpK14 significantly reduced the minimum inhibitory concentrations (MICs) of β-lactams and had no effect on non-β-lactams against VRSA and VRE. MICs of CSpK14/oxacillin and CSpK14/ampicillin were reduced by 8- to 64-fold and 2- to 16-fold, respectively. The time killing assay between CSpK14/oxacillin (2.29–2.37 Δlog₁₀CFU/mL at 24 h) and CSpK14/ampicillin (2.30–2.38 Δlog₁₀CFU/mL at 24 h) being >2-fold and fractional inhibitory concentration index ?0.5 revealed synergy. Furthermore, the biofilms formed by VRSA and VRE were reduced completely. CSpK14 was simple to purify, had low molecular mass, was stable over a wide pH range or tested chemicals, had broad inhibitory spectrum, and possessed potent synergistic antimicrobial-antibiofilm properties. CSpK14 synergistically enhanced the efficacy of β-lactams and is therefore suitable for combination therapy.     

Bacteriocin production by lactic acid bacteria isolated from Rioja red wines

Forty-two lactic acid bacteria (LAB) of the genera Lactobacillus (32), Leuconostoc (6), Pediococcus (3) and Lactococcus (1), isolated from Rioja red wines, were tested for antimicrobial activity. All these strains, as well as 18 Leuconostoc oenos and 19 yeast strains were used as indicators. Only nine strains showed antimicrobial activity, and all were of the species Lactobacillus plantarum, which constitutes the predominant microflora in Rioja red wines after alcoholic fermentation. Lact. plantarum strain J-51 showed the widest range of action, inhibiting the growth of 31 strains of the four studied LAB genera. Lact. plantarum J-51 antimicrobial activity was lost after treatment with proteases, suggesting a proteinaceous nature for this activity. It was found to be stable between pH 3 and 9 and under strong heating conditions (100 degrees C for 60 min). Polymerase chain reaction (PCR) analysis of Lact. plantarum J-51 genome revealed the presence of the plnA gene that encodes the plantaricin precursor PlnA. A 366-bp fragment was sequenced and showed 95% identity with pln locus of Lact. plantarum C-11. The deduced precursor peptide sequence showed one mutation (Gly7 to Ser7) at the double glycine leader peptide, and the three putative 26-, 23- and 22-residue active peptides remain identical to those of Lact. plantarum C-11. Therefore, antimicrobial peptides constitute a potent adaptation advantage for those strains that dominate in a medium such as wine, and can play an important role in the ecology of wine microflora.     

肾综合征出血热病毒LR1株在Vero细胞上适应的特性研究

选取不同代次的ＬＲ１毒株在Ｖｅｒｏ细胞上传代适应,不同天数连续动态观察细胞病变情况,同时用免疫荧光法和ＥＬＩＳＡ进行抗原检测,收毒前细胞反复冻融及超声波破碎,细胞破碎前后用ＥＬＩＳＡ检测抗原含量,半微量空斑法检测病毒滴度。结果证明：ＬＲ１株病毒能在Ｖｅｒｏ细胞上产生细胞病变,病变程度与其毒力明显相关;ＬＲ１株病毒在Ｖｅｒｏ细胞上繁殖的最佳收毒时间为１１天左右;病毒释放性较差,冻融和超声波破碎细胞能明显提高其抗原含量和病毒滴度     

Purification and partial amino acid sequence of plantaricin S, a bacteriocin produced by Lactobacillus plantarum LPCO10, the activity of which depends on the complementary action of two peptides.

Plantaricin S, one of the two bacteriocins produced by Lactobacillus plantarum LPCO10, which was isolated from a green-olive fermentation (R. Jiménez-Díaz, R.M. Ríos-Sánchez, M. Desmazeaud, J.L.Ruiz-Barba, and J.-C. Piard, Appl. Environ. Microbiol. 59:1416-1424, 1993), has been purified to homogeneity by ammonium sulfate precipitation, by binding to SP-Sepharose fast-flow, phenyl-Sepharose CL-4B, and C2/C18 reverse-phase chromatographies. The purification resulted in a final yield of 91.6% and a 352,617-fold increase in the specific activity. The bacteriocin activity was associated with two distinct peptides, termed alpha and beta, which were separated by C2/C18 reverse-phase chromatography. Although beta alone appeared to retain a trace of inhibitory activity, the complementary action of both the alpha and beta peptides was required for full bacteriocin activity, as judged by both the agar well diffusion and the microtiter plate assays. From the N-terminal end, 26 and 24 amino acids residues of alpha and beta, respectively, were sequenced. Further attempts at sequencing revealed no additional amino acids residues, suggesting that either modifications in the next amino acid residue blocked the sequencing region or that the C-terminal end had been reached. The amino acid sequences of alpha and beta show no apparent homology to each or to other bacteriocins purified from lactic acid bacteria.     

Two novel cationic antifungal peptides isolated from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> HN-10 and their inhibitory activity against <Emphasis Type="Italic">Trichothecium roseum</Emphasis>

Public concern for food safety and environmental issues and the increase in fungicide-resistant pathogen have enhanced the interest in developing alternative methods to fungicides to control postharvest fruit decay. In this study, a bacterial strain isolated from stale potato vermicelli was identified as Bacillus pumilus HN-10 based on morphological characteristics and 16S rRNA gene sequence analysis. Furthermore, two novel cationic antifungal peptides named P-1 and P-2 were purified from B. pumilus HN-10 using macroporous adsorbent resin AB-8, Sephadex G-100 chromatography, and reversed-phase high-performance liquid chromatography. The primary structure of P-1 and P-2, which were proved to be novel antifungal peptides by BLAST search in NCBI database, was PLSSPATLNSR and GGSGGGSSGGSIGGR with a molecular weight of 1142.28 and 1149.14 Da, respectively, as indicated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Both P-1 and P-2 exhibited strong antifungal activity against Trichothecium roseum with minimum inhibitory concentrations starting from 1 μg/mL. The two novel antifungal peptides were stable below 80 °C for 2 h, but lost their activity in 15 min at 121 °C. In addition, they were resistant to the proteolytic action of pepsin, trypsin, and papain, and stable within a wide range of pH (2.0–12.0). These results showed that P-1 and P-2 are novel cationic antifungal peptides with specific activity against T. roseum.     

苏芸金杆菌广谱杀虫Tml3—14菌株晶体毒素及毒力特性

以新筛选的对鞘翅目,鳞翊目、双翅目均具毒性的B.t.Tml3-14菌株为材料,用HD-1、3370-1为参考菌株,比较了伴孢晶体蛋白的多肽组成,以及经三种昆虫肠道酶降解产生的抗酶多肽组分。SDS-PAGE分析表明：Tml3-14晶体蛋白含1 38kD、l 32kD主要成分和65kD次要我分。其晶体分别由三种昆虫肠道酶消化产生的毒性多肽,经生物测定,杀家蚕的毒性成分为68.5kD、59kD多肽;杀斜纹夜蛾毒性成分为71kD,67kD和59.6kD多肽;杀马铃薯瓢虫毒性成分为69kD、65kD多肽。它与HD01、3370-1晶体均有明显差异。     

Purification and genetic characterization of plantaricin NC8, a novel coculture-inducible two-peptide bacteriocin from Lactobacillus plantarum NC8

A new, coculture-inducible two-peptide bacteriocin named plantaricin NC8 (PLNC8) was isolated from Lactobacillus plantarum NC8 cultures which had been induced with Lactococcus lactis MG1363 or Pediococcus pentosaceus FBB63. This bacteriocin consists of two distinct peptides, named alpha and beta, which were separated by C(2)-C(18) reverse-phase chromatography and whose complementary action is necessary for full plantaricin NC8 activity. N-terminal sequencing of both purified peptides showed 28 and 34 amino acids residues for PLNC8 alpha and PLNC8 beta, respectively, which showed no sequence similarity to other known bacteriocins. Mass spectrometry analysis showed molecular masses of 3,587 Da (alpha) and 4,000 Da (beta). The corresponding genes, designated plNC8A and plNC8B, were sequenced, and their nucleotide sequences revealed that both peptides are produced as bacteriocin precursors of 47 and 55 amino acids, respectively, which include N-terminal leader sequences of the double-glycine type. The mature alpha and beta peptides contain 29 and 34 amino acids, respectively. An open reading frame, orfC, which encodes a putative immunity protein was found downstream of plNC8B and overlapping plNC8A. Upstream of the putative -35 region of plNC8B, two direct repeats of 9 bp were identified, which agrees with the consensus sequence and structure of promoters of class II bacteriocin operons whose expression is dependent on an autoinduction mechanism.     

The effects of the cyanobacterium Microcystis aeruginosa, the cyanobacterial hepatotoxin microcystin–LR, and ammonia on growth rate and ionic regulation of brown trout

Brown trout were exposed for 63 days to five treatments: a control; the purified cyanobacterial hepatotoxin microcystin—LR (MC—LR) (41—57 μg MC—LR 1^?1); lysed toxic Microcystis aeruginosa cells (41–68 μg MC—LR 1^?1 and 288 μg chlorophyll a 1^?1); lysed non—toxic M. aeruginosa cells (non—MC—LR containing and 288 μg chlorophyll a 1^?1); ammonia (65–325 μg NH₃ 1^?1). All treatments produced significantly reduced growth compared to controls (P<0·05, Fisher test). Exposure to ammonia resulted weight loss over the first 7 days followed by weight increase, though at a significantly lower level than in the other treatments. First exposed to lysed toxic M. aeruginosa cells grew less than those exposed to lysed non—toxic cyanobacteria or purified MC—LR. Sodium influx rates after 63 days exposure to purified MC—LR, lysed toxic M. aeruginosa cells, or ammonia showed a significant increase compared to control fish or those exposed to lysed non—toxic M. aeruginosa cells. There were no significant differences in Na⁺ efflux or net Na⁺ uptake rates between treatments. Significant increases in body Na⁺ and Cl^— were seen in fish exposed to lysed toxic M. aeruginosa cells or ammonia. Only fish exposed to ammonia showed a significant increase in body ammonia. Short—term exposure, over 4 h, to lysed toxic cells, non—toxic cells or purified MC—LR resulted in insignificant changes in Na⁺ flux rates compared to controls although there was a significant net Na⁺ loss in fish exposed to ammonia. Chronic exposure of fish to toxic cyanobacterial blooms may result in ionic imbalance and reduced growth.     

The V alpha 14 NKT cell TCR exhibits high-affinity binding to a glycolipid/CD1d complex

Most CD1d-dependent NKT cells in mice have a canonical V alpha 14J alpha 18 TCR rearrangement. However, relatively little is known concerning the molecular basis for their reactivity to glycolipid Ags presented by CD1d. Using glycolipid Ags, soluble forms of a V alpha 14 NKT cell-derived TCR, and mutant and wild-type CD1d molecules, we probed the TCR/CD1d interaction by surface plasmon resonance, tetramer equilibrium staining, and tetramer staining decay experiments. By these methods, several CD1d alpha-helical amino acids could be defined that do not greatly alter lipid binding, but that affect the interaction with the TCR. Binding of the V alpha 14(+) TCR to CD1d requires the agonist alpha-galactosylceramide (alpha-GalCer), as opposed to the nonantigenic beta-galactosylceramide, although both Ags bind to CD1d, indicating that the carbohydrate moiety of the CD1d-bound Ag plays a major role in the TCR interaction. The TCR has a relatively high-affinity binding to the alpha-GalCer/CD1d complex, with a particularly slow off rate. These unique properties are consistent with the coreceptor-independent action of the V alpha 14 TCR and may be related to the intense response to alpha-GalCer by NKT cells in vivo.     

Characterization and purification of a new bacteriocin with a broad inhibitory spectrum produced by Lactobacillus plantarum lp 31 strain isolated from dry-fermented sausage

Aims:  Characterization and purification of a new bacteriocin produced by Lactobacillus plantarum LP 31 strain, isolated from Argentinian dry-fermented sausage.
Methods and Results:  Lactobacillus plantarum LP 31 strain produces an antimicrobial compound that inhibits the growth of food-borne pathogenic bacteria. It was inactivated by proteolytic enzymes, was stable to heat and catalase and exhibited maximum activity in the pH range from 5·0 to 6·0. Consequently, it was characterized as a bacteriocin. It was purified by RP (reverse-phase) solid-phase extraction, gel filtration chromatography and RP-HPLC. Plantaricin produced by Lact. plantarum LP 31 is a peptide with a molecular weight of 1558·85 Da as determined by Maldi-Tof mass spectrometry and contains 14 amino acid residues. It was shown to have a bactericidal effect against Pseudomonas sp., Staphylococcus aureus , Bacillus cereus and Listeria monocytogenes.
Conclusions:  The bacteriocin produced by Lact. plantarum LP 31 may be considered as a new plantaricin according to its low molecular weight and particular amino acid composition.
Significance and Impact of the Study:  In view of the interesting inhibitory spectrum of this bacteriocin and because of its good technological properties (resistance to heat and activity at acidic pH), this bacteriocin has potential applications as a biopreservative to prevent the growth of food-borne pathogens and food spoilage bacteria in certain food products.     

A model of activation of the protein tyrosine phosphatase SHP-2 by the human leptin receptor

Signalling through the leptin receptor has been shown to activate the SH2 domain-containing tyrosine phosphatase SHP-2 through tyrosine phosphorylation. The human leptin receptor contains five tyrosine residues in the cytoplasmic domain that may become phosphorylated. We show here using BIAcore studies, wherein binding of peptides to SHP-2 was detected, that peptides corresponding to sequences containing phosphotyrosines 974 and 986 (LR974P and LR986P, respectively) from the leptin receptor cytoplasmic domain were the only two peptides that bound to the enzyme. Binding of LR974P to SHP-2 was inhibited in a dose-dependent fashion by orthovanadate, whereas binding of LY986P was not, indicating that the enzyme binds to these peptides through different sites. Only the leptin receptor-derived peptide corresponding to tyrosine 974 was dephosphorylated by recombinant purified SHP-2. Time courses of the reaction were complex, and fitted a two exponent rate equation. Preincubation of SHP-2 with LR986P markedly activated the enzyme at early time points and time courses of the activated enzyme fitted a single exponential first order rate equation. We propose that LR974P binds to the active site of SHP-2, whereas LR986P may bind to the N- and C-terminal SH2 domains of SHP-2, thus activating the phosphatase activity. These data support a model in which SHP-2 binds to phosphotyrosine 986 in the activated leptin receptor and is activated to dephosphorylate phosphotyrosine 974, downregulating signalling events emanating from SH2 domain-containing proteins that bind here.     

The effects of microcystin‐LR in Oryza sativa root cells: F‐actin as a new target of cyanobacterial toxicity

• Microcystins are toxins produced by cyanobacteria, notorious for negatively affecting a wide range of living organisms, among which several plant species. Although microtubules are a well‐established target of microcystin toxicity, its effect on filamentous actin (F‐actin) in plant cells has not yet been studied.
• Τhe effects of microcystin‐LR (MC‐LR) and an extract of a microcystin‐producing freshwater cyanobacterial strain (Microcystis flos‐aquae TAU‐MAC 1510) on the cytoskeleton (F‐actin and microtubules) of Oryza sativa (rice) root cells were studied with light, confocal, and transmission electron microscopy. Considering the role of F‐actin in endomembrane system distribution, the endoplasmic reticulum and the Golgi apparatus in extract‐treated cells were also examined.
• F‐actin in both MC‐LR- and extract‐treated meristematic and differentiating root cells exhibited time‐dependent alterations, ranging from disorientation and bundling to the formation of ring‐like structures, eventually resulting in a collapse of the F‐actin network after longer treatments. Disorganization and eventual depolymerization of microtubules, as well as abnormal chromatin condensation were observed following treatment with the extract, effects which could be attributed to microcystins and other bioactive compounds. Moreover, cell cycle progression was inhibited in extract‐treated roots, specifically affecting the mitotic events. As a consequence of F‐actin network disorganization, endoplasmic reticulum elements appeared stacked and diminished, while Golgi dictyosomes appeared aggregated.
• These results support that F‐actin is a prominent target of MC‐LR, both in pure form and as an extract ingredient. Endomembrane system alterations can also be attributed to the effects of cyanobacterial bioactive compounds (including microcystins) on the F‐actin cytoskeleton.

     

Laminin Receptor 37/67LR Regulates Adhesion and Proliferation of Normal Human Intestinal Epithelial Cells

Interactions between the cell basal membrane domain and the basement membrane are involved in several cell functions including proliferation, migration and differentiation. Intestinal epithelial cells can interact with laminin, a major intestinal basement membrane glycoprotein, via several cell-surface laminin-binding proteins including integrin and non-integrin receptors. The 37/67kDa laminin receptor (37/67LR) is one of these but its role in normal epithelial cells is still unknown. The aim of this study was to characterise the expression pattern and determine the main function of 37/67LR in the normal human small intestinal epithelium. Immunolocalization studies revealed that 37/67LR was predominantly present in the undifferentiated/proliferative region of the human intestinal crypt in both the immature and adult intestine. Using a human intestinal epithelial crypt (HIEC) cell line as experimental model, we determined that 37/67LR was expressed in proliferative cells in both the cytoplasmic and membrane compartments. Small-interfering RNA-mediated reduction of 37/67LR expression led to HIEC cell-cycle reduction and loss of the ability to adhere to laminin-related peptides under conditions not altering ribosomal function. Taken together, these findings indicate that 37/67LR regulates proliferation and adhesion in normal intestinal epithelial cells independently of its known association with ribosomal function.     

Knock-Down of the 37kDa/67kDa Laminin Receptor LRP/LR Impedes Telomerase Activity

Cancer has become a major problem worldwide due to its increasing incidence and mortality rates. Both the 37kDa/67kDa laminin receptor (LRP/LR) and telomerase are overexpressed in cancer cells. LRP/LR enhances the invasiveness of cancer cells thereby promoting metastasis, supporting angiogenesis and hampering apoptosis. An essential component of telomerase, hTERT is overexpressed in 85–90% of most cancers. hTERT expression and increased telomerase activity are associated with tumor progression. As LRP/LR and hTERT both play a role in cancer progression, we investigated a possible correlation between LRP/LR and telomerase. LRP/LR and hTERT co-localized in the perinuclear compartment of tumorigenic breast cancer (MDA_MB231) cells and non-tumorigenic human embryonic kidney (HEK293) cells. FLAG^® Co-immunoprecipitation assays confirmed an interaction between LRP/LR and hTERT. In addition, flow cytometry revealed that both cell lines displayed high cell surface and intracellular LRP/LR and hTERT levels. Knock-down of LRP/LR by RNAi technology significantly reduced telomerase activity. These results suggest for the first time a novel function of LRP/LR in contributing to telomerase activity. siRNAs targeting LRP/LR may act as a potential alternative therapeutic tool for cancer treatment by (i) blocking metastasis (ii) promoting angiogenesis (iii) inducing apoptosis and (iv) impeding telomerase activity.     

Anti-LRP/LR Antibody W3 Hampers Peripheral PrPSc Propagation in Scrapie Infected Mice

We identified the 37kDa/67kDa laminin receptor (LRP/LR) as a cell surface receptor for the cellular prion protein (PrP^c) and the infectious prion protein (PrP^Sc). Recently, we showed that anti-LRP/LR antibody W3 cured scrapie infected N2a cells. Here, we demonstrate that W3 delivered by passive immunotransfer into C57BL/6 mice reduced the PrP^Sc content in the spleen significantly by 66%, demonstrating an impairment of the peripheral PrP^Sc propagation. In addition, we observed a 1.8-fold increase in survival of anti-LRP/LR antibody W3 treated mice (mean survival of 31 days) compared to preimmune serum treated control animals (mean survival of 17 days). We conclude that the significant effect of anti-LRP/LR antibody W3 on the reduction of peripheral PrP^Sc propagation might be due to the blockage of the prion receptor LRP/LR which is required, as previously shown in vitro, for PrP^Sc propagation in vivo.Key Words: 37kDa/67kDa laminin receptor, LRP/LR, prion, PrP, TSE-therapy     

Anti-LRP/LR Specific Antibody IgG1-iS18 Impedes Adhesion and Invasion of Liver Cancer Cells

Two key events, namely adhesion and invasion, are pivotal to the occurrence of metastasis. Importantly, the 37 kDa/67 kDa laminin receptor (LRP/LR) has been implicated in enhancing these two events thus facilitating cancer progression. In the current study, the role of LRP/LR in the adhesion and invasion of liver cancer (HUH-7) and leukaemia (K562) cells was investigated. Flow cytometry revealed that the HUH-7 cells displayed significantly higher cell surface LRP/LR levels compared to the poorly-invasive breast cancer (MCF-7) control cells, whilst the K562 cells displayed significantly lower cell surface LRP/LR levels in comparison to the MCF-7 control cells. However, Western blotting and densitometric analysis revealed that all three tumorigenic cell lines did not differ significantly with regards to total LRP/LR levels. Furthermore, treatment of liver cancer cells with anti-LRP/LR specific antibody IgG1-iS18 (0.2 mg/ml) significantly reduced the adhesive potential of cells to laminin-1 and the invasive potential of cells through the ECM-like Matrigel, whilst leukaemia cells showed no significant differences in both instances. Additionally, Pearson''s correlation coefficients suggested direct proportionality between cell surface LRP/LR levels and the adhesive and invasive potential of liver cancer and leukaemia cells. These findings suggest the potential use of anti-LRP/LR specific antibody IgG1-iS18 as an alternative therapeutic tool for metastatic liver cancer through impediment of the LRP/LR- laminin-1 interaction.