A Peptide from the Beta-strand Region of CD2 Protein that Inhibits Cell Adhesion and Suppresses Arthritis in a Mouse Model

Cell adhesion molecules play a central role at every step of the immune response. The function of leukocytes can be regulated by modulating adhesion interactions between cell adhesion molecules to develop therapeutic agents against autoimmune diseases. Among the different cell adhesion molecules that participate in the immunologic response, CD2 and its ligand CD58 (LFA-3) are two of the best-characterized adhesion molecules mediating the immune response. To modulate the cell adhesion interaction, peptides were designed from the discontinuous epitopes of the β-strand region of CD2 protein. The two strands were linked by a peptide bond. β-Strands in the peptides were nucleated by inserting a β-sheet-inducing Pro-Gly sequence with key amino acid sequences from CD2 protein that binds to CD58. Using a fluorescence assay, peptides that exhibited potential inhibitory activity in cell adhesion were evaluated for their ability to bind to CD58 protein. A model for peptide binding to CD58 protein was proposed based on docking studies. Administration of one of the peptides, P3 in collagen-induced arthritis in the mouse model, indicated that peptide P3 was able to suppress rheumatoid arthritis in mice.     

Pulmonary delivery of elcatonin using surface-modified liposomes to improve systemic absorption: Polyvinyl alcohol with a hydrophobic anchor and chitosan oligosaccharide as effective surface modifiers

The aim of this study was to investigate the feasibility of surface-modified liposomes for pulmonary delivery of a peptide. Chitosan oligosaccharide (oligoCS) and polyvinyl alcohol with a hydrophobic anchor (PVA-R) were used as surface modifiers. The effect of liposomal surface modification on the behavior of the liposomes on pulmonary administration and potential toxicity were evaluated in vitro and in vivo. In an association study with A549 cells, PVA-R modification reduced interaction with A549 cells, whereas oligoCS modification electrostatically enhanced cellular interaction. The therapeutic efficacy of elcatonin (eCT) after pulmonary administration to rats was significantly enhanced and prolonged for 48 h after separate administration with oligoCS- or PVA-R-modified liposomes. oligoCS-modified liposomes adhered to lung tissues and caused opening of tight junctions, which enhanced eCT absorption. On the other hand, PVA-R-modified liposomes induced long-term retention of eCT in the lung fluid, leading to sustained absorption. Consequently, surface modification of liposomes with oligoCS or PVA-R has potential for effective peptide drug delivery through pulmonary administration.     

Discovery of Trp-Nle-Tyr-Met as a novel agonist for human formyl peptide receptor-like 1

Formyl peptide receptor-like 1 (FPRL1) is a structural homologue of FPR, which binds chemotactic peptides as small as three amino acids (e.g., fMet-Leu-Phe, fMLF) and activates potent bactericidal functions in neutrophils. In comparison, FPRL1 ligands include peptides of 6-104 amino acids, such as Trp-Lys-Tyr-Met-Val-[d]Met (WKYMVm) and other synthetic peptides. To determine the core peptide sequence required for FPRL1 activation, we prepared various analogues based on WKYMVm and evaluated their bioactivities in an FPRL1-transfected cell line. Although substitution of d-Met(6) resulted in loss of activity, removal of Val(5) together with d-Met(6) produced a peptide that retained most of the bioactivities of the parent peptide. The resulting peptide, WKYM, represents a core structure for an FPRL1 ligand. Further substitution of Lys(2) with Nle slightly improved the potency of the tetrapeptide, which selectively activates FPRL1 over FPR. Based on these structure-activity relationship studies, we propose a model in which the modified tetrapeptide Trp-Nle-Tyr-Met (WNleYM) binds to FPRL1 through aromatic interactions involving the side chains of Trp(1) and Tyr(3), hydrophobic interaction of Nle(2), and the thio-based hydrogen bonding of Met(4), with the respective residues in FPRL1 which have not been identified. The identification of the core sequence of a potent peptide agonist provides a structural basis for future design of peptidomimetics as potential therapeutic agents for FPRL1-related disorders.     

The NFL-TBS.40-63 anti-glioblastoma peptide enters selectively in glioma cells by endocytosis

Glioblastoma are the most frequent and aggressive tumour of the nervous system despite surgical resection associated with chemotherapy and radiotherapy. Recently, we showed that the NFL-TBS.40-63 peptide corresponding to the sequence of a tubulin-binding site of neurofilaments, enters selectively in glioblastoma cells where it blocks microtubule polymerization, inhibits their proliferation, and reduces tumour development in rats bearing glioblastoma (0050 and 0040). Here, we characterized the molecular mechanism responsible for the uptake of NFL-TBS.40-63 peptide by glioblastoma cells. Unlike other cell penetrating peptides (CPPs), which use a balance between endocytosis and direct translocation, the NFL-TBS.40-63 peptide is unable to translocate directly through the membrane when incubated with giant plasma membrane vesicles. Then, using a panel of markers and inhibitors, flow cytometry and confocal microscopy investigations showed that the uptake occurs mainly through endocytosis. Moreover, glycosaminoglycans and αVβ3 integrins are not involved in the NFL-TBS.40-63 peptide recognition and internalization by glioblastoma cells. Finally, the signalling of tyrosine kinase receptors is involved in the peptide uptake, especially via EGFR overexpressed in tumour cells, indicating that the uptake of NFL-TBS.40-63 peptide by glioblastoma cells is related to their abnormally high proliferative activity.     

Ceramides and cerebrosides from the marine bryozoan Bugula neritina inhabiting South China Sea

From the marine bryozoan Bugula neritina inhabiting South China Sea, a new ceramide named (2S,3R,4E)-2-(14′-methyl-pentadecanoylamino)-4-octadecene-l,3-diol (1) and a new cerebroside named 1-O-(β-d-glucopyranosyl)-(2S,3R,4E)-2-(heptadecanoylamino)-4-octadecene-l,3-diol (6), together with one known ceramide (2) and three known cerebrosides (3, 4, and 5), were isolated. Their structures were deduced by extensive spectral analysis and chemical evidences. Compound 1 is branched with a methyl [–CH(CH₃)₂] in the fatty acid moiety, which is a rare structural feature among ceramides. Compound 6 is a new cerebroside with 17 carbons in the fatty acid moiety, while 5 is a new natural product which was isolated from a natural origin for the first time.     

Cellular Uptake But Low Permeation of Human Calcitonin-Derived Cell Penetrating Peptides and Tat(47-57) Through Well-Differentiated Epithelial Models

PURPOSE: To investigate whether cell penetrating peptides (CPP) derived from human calcitonin (hCT) possess, in addition to cellular uptake, the capacity to deliver their cargo through epithelial barriers. METHODS: Cellular uptake of hCT(9-32) and permeation of six hCT-derived peptides, namely, hCT(9-32), hCT(12-32), hCT(15-32), hCT(18-32), hCT(21-32), and a random sequence of hCT(9-32) were evaluated in fully organized confluent Madin-Darby canine kidney (MDCK), Calu-3, and TR146 cell culture models. For comparison, Tat(47-57) and penetratin(43-58) were investigated. The peptides were N-terminally labeled with carboxyfluorescein (CF). Uptake in the well-differentiated epithelial models was observed by confocal laser scanning microscopy (CLSM), whereas permeation through the models was analyzed by reversed-phase (RP)-HPLC. RESULTS: In MDCK epithelium hCT(9-32), Tat(47-57) and penetratin(43-58) demonstrated punctuated cytoplasmic distribution. In Calu-3, Tat(47-57) and penetratin(43-58) were simultaneously localized in a punctuated cytoplasmic and paracellular distribution, whereas hCT(9-32) showed strict paracellular distribution. By contrast, in TR146 cells, Tat(47-57) was located strictly paracellularily, whereas penetratin(43-58) showed a punctuated cytoplasmic pattern and hCT(9-32) both. The transepithelial permeability of all tested peptides and their cargo was lower than that of paracellular markers. CONCLUSIONS: The CPP uptake pattern depends on both the type of peptide and the cell culture model. In general, the investigated CPP have no apparent potential for systemic drug delivery across epithelia. Nevertheless, distinct patterns of cellular distribution may offer a potential for localized epithelial delivery.     

3'-Azido-2',3'-dideoxythymidine induced deficiency of thymidine kinases 1, 2 and deoxycytidine kinase in H9 T-lymphoid cells

Continuous cultivation of T-lymphoid H9 cells in the presence of 3′-azido-2′,3′-dideoxythymidine (AZT) resulted in a cell variant cross-resistant to both thymidine and deoxycytidine analogs. Cytotoxic effects of AZT, 2′,3′-didehydro-3′-deoxythymidine as well as different deoxycytidine analogs such as 2′,3′-dideoxycytidine, 2′,2′-difluoro-2′-deoxycytidine (dFdC) and 1-ß-D-arabinofuranosylcytosine (Ara-C) were strongly reduced in H9 cells continuously exposed to AZT when compared to parental cells (>8.3-, >6.6-, >9.1-, 5×10⁴-, 5×10³-fold, respectively). Moreover, anti-HIV-1 effects of AZT, d4T, ddC and 2′,3′-dideoxy-3′-thiacytidine (3TC) were significantly diminished (>222-, >25-, >400-, >200-fold, respectively) in AZT-resistant H9 cells. Study of cellular mechanisms responsible for cross-resistance to pyrimidine analogs in AZT-resistant H9 cells revealed decreased mRNA levels of thymidine kinase 1 (TK1) and lack of deoxycytidine kinase (dCK) mRNA expression. The loss of dCK gene expression was confirmed by western blot analysis of dCK protein as well as dCK enzyme activity assay. Moreover, enzyme activity of TK1 and TK2 was reduced in AZT-resistant cells. In order to determine whether lack of dCK affected the formation of the active triphosphate of the deoxycytidine analog dFdC, dFdCTP accumulation and retention was measured in H9 parental and AZT-resistant cells after exposure to 1 and 10 μM dFdC. Parental H9 cells accumulated about 30 and 100 pmol dFdCTP/10⁶ cells after 4 hr, whereas in AZT-resistant cells no dFdCTP accumulation was detected. These results demonstrate that continuous treatment of H9 cells in the presence of AZT selected for a thymidine analog resistant cell variant with cross-resistance to deoxycytidine analogs, due to deficiency in TK1, TK2, and dCK.     

EGCG-meditated cyto- and genotoxicity in HaCat keratinocytes is impaired by cell-mediated clearance of auto-oxidation-derived H2O2: an algorithm for experimental setting correction

Several lines of evidence suggest that besides antioxidant also prooxidant properties are crucially involved in cytotoxic and protective activities of the major green tea catechin epigallocatechin-3-gallate (EGCG) in vitro (Elbling et al., 2011). Furthermore recent data suggest that EGCG induces oxidative stress also in vivo (Li et al., 2010). Here we set out to identify factors modulating cellular effects of EGCG in vitro. Using the HaCat keratinocytes model, we demonstrate that the cytotoxic, genotoxic and signal-activating effects of EGCG are significantly dependent on the ratio of cell number to working volume. Treatment with identical EGCG concentrations at altered experimental settings resulted in IC₅₀ values differing up to orders of magnitude and could even exert contradictory effects. This effect was based on cell-mediated clearance of autooxidation-derived H₂O₂ from the supernatant. In order to estimate EGCG/H₂O₂ concentrations equally effective under different settings, we have rationally derived and experimentally verified a simple algorithm relating concentration, working volume, cell number and - indirectly - exposure time. Algorithm application resulted in similar H₂O₂ clearance curves from cell supernatants as well as comparable EGCG/H₂O₂ effects at different settings. Our results demonstrate the importance of standardized experimental settings when investigating cytotoxic and/or beneficial effects of autooxidizing compounds.     

Structure–activity relationship of truncated and substituted analogues of the intracellular delivery vector Penetratin

Abstract: Peptides derived from the third α‐helix of the homeodomain (residues 43–58; Penetratin) of Antennapedia, a Drosophila homeoprotein, were prepared by simultaneous multiple synthesis. Sets of N‐ and C‐terminally truncated peptides, as well as a series of alanine substitution analogues, were studied. Cell penetration assays using human cell cultures with these peptides revealed that the C‐terminal segment ⁵²Arg‐Arg‐Met‐Lys‐Trp‐Lys‐Lys⁵⁸ of the parent sequence was necessary and sufficient for efficient cell membrane translocation. Individual Ala substitutions of the peptide’s basic residues led to markedly decreased cell internalization ability, whereas replacement of hydrophobic residues was tolerated surprisingly well. Subcellular localization was seen to be affected by substitutions, with analogues being addressed preferentially to the cytosol or to the nucleus. Conformational constriction of the Penetratin sequence through placement and oxidation of flanking cysteine residues afforded a cyclic disulfide peptide which had lost most of its membrane translocation capacity.     

Irreversible binding of a novel phenylisothiocyanate tropane analog to monoamine transporters in rat brain

Irreversible tropane analogs have been useful in identifying binding sites of cocaine on biogenic amine transporters, including transporters for dopamine (DAT), serotonin (SERT) and norepinephrine (NET). The present study characterizes the properties of the novel phenylisothiocyanate tropane HD-205, synthesized from the highly potent 2-napthyl tropane analog WF-23. In radioligand binding studies in brain membranes, direct IC(50) values of HD-205 were 4.1, 14 and 280nM at DAT, SERT and NET, respectively. Wash-resistant binding was characterized by preincubation of HD-205 with brain membranes, followed by extensive washing before performing transporter radioligand binding. Results for HD-205 showed wash-resistant IC(50) values of 191, 230 and 840nM at DAT, SERT and NET, respectively. Saturation binding studies with [(125)I]RTI-55 in membranes pretreated with 100nM HD-205 showed that HD-205 significantly decreased the B(max) but not K(D) of DAT and SERT binding. To further characterize its irreversible binding, an iodinated analog of HD-205, HD-244, was prepared from a trimethylsilyl precursor. The direct IC(50) of HD-244 at DAT was 20nM. [(125)I]HD-244 was synthesized with chloramine-T, purified on HPLC, reacted with rat striatal membranes, and proteins were separated by SDS-PAGE. Results showed several non-specific labeled bands, but only a single specific band of radioactivity co-migrating with an immunoreactive DAT band at approx. 80 kilodaltons was detected, suggesting that [(125)I]HD-244 covalently labeled DAT protein in striatal membranes. These results demonstrate that phenylisothiocyanate analogs of WF-23 can be used as potential ligands to map distinct binding sites of cocaine analogs at DAT.     

Novel chemoattractant peptides for human leukocytes

Phospholipase A(2) plays a key role in phagocytic cell functions. By screening a synthetic hexapeptide combinatorial library, we identified 24 novel peptides based on their ability to stimulate arachidonic acid release associated with cytosolic phospholipase A(2) activity in differentiated HL60 cells. The identified peptides, that contain the consensus sequence (K/R/M)KYY(P/V/Y)M, also induce intracellular calcium release in a pertussis toxin-sensitive manner showing specific action on phagocytic leukocytes, but not on other cells. Functionally, the peptides stimulate superoxide generation and chemotactic migration in human neutrophils and monocytes. Four of the tested active peptides were ligands for formyl peptide receptor like 1. Among these, two peptides with the consensus sequence (R/M)KYYYM can induce intracellular calcium release in undifferentiated HL60 cells that do not express formyl peptide receptor like 1, indicating usage of other receptor(s). A study of intracellular signaling in differentiated HL60 cells induced by the peptides has revealed that four of the novel peptides can induce extracellular signal-regulated protein kinase activation via shared and distinct signaling pathways, based on their dependence of phospatidylinositol-3-kinase, protein kinase C, and MEK. These peptides provide previously unavailable tools for study of differential signaling in leukocytes.     

Neuroprotective effects of TEMPOL in central and peripheral nervous system models of Parkinson's disease

TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) is a stable nitroxyl antioxidant. Previous studies have suggested that TEMPOL is protective in acute disorders thought to involve reactive oxygen species (ROS), such as ischemic stroke and cardiac reperfusion injury. Oxidized TEMPOL can be recycled to its redox-active reducing form by co-administration with polynitroxylated albumin, making it a candidate as a pharmacological "reservoir" for reducing potential of use in chronic disorders involving ROS. The present studies examine the efficacy of TEMPOL in cell culture and animal models of the central and peripheral dysfunction associated with Parkinson's disease, a disorder in the pathogenesis of which ROS generated from dopamine have been implicated. Antioxidants have been proposed as both preventive and symptomatic therapy for Parkinson's disease. TEMPOL protects MN9D dopaminergic mesencephalic cells in culture from 6-hydroxydopamine (6-OHDA)-induced apoptosis. Translocation of the p65 component of NF-kappaB to the nucleus accompanies protection by TEMPOL. In vivo, intraperitoneal TEMPOL protects mice from intrastriatal 6-OHDA-induced cell and dopamine metabolite loss in the striatum. TEMPOL also protects mice against the 6-OHDA-induced rotational behavior elicited by intrastriatal administration of d-amphetamine. In addition, TEMPOL protects mice from the ptosis, activity level decrement, and mortality induced by intraperitoneal administration of 6-OHDA, a model of autonomic dysfunction in Parkinson's disease. Adjunctive use of polynitroxylated albumin enhances the in vitro and in vivo effects of TEMPOL.     

The Proteolytic Stability and Cytotoxicity Studies of l‐Aspartic Acid and l‐Diaminopropionic Acid derived β‐Peptides and a Mixed α/β‐Peptide

The use of peptides as drugs in pharmaceutical applications is hindered by their susceptibility to proteolysis and therefore low bioavailability. β‐Peptides that contain an additional methylene group in the backbone, are gaining recognition from a pharmaceutical stand point as they are considerably more resilient to proteolysis and metabolism. Recently, we reported two new classes of β ‐peptides, β ³‐ and β²‐peptides derived from l ‐aspartic acid and l ‐diaminopropionic acid, respectively. Here, we report the proteolytic stability of these β‐peptidic compounds and a mixed α /β‐peptide against three enzymes (pronase, trypsin and elastase), as well as, human serum. The stability of these peptides was compared to an α‐peptide. Peptides containing β‐linkages were resistant to all conditions. The mixed α /β‐peptide, however, exhibited proteolysis in the presence of trypsin and pronase but not elastase. The rate of degradation of the mixed α /β‐peptide was slower than that would be expected for an α‐peptide. In addition, these β‐peptides were not toxic to HeLa and COS‐1 cell lines as observed by MTT cytotoxicity assay. These results expand the scope of mixed α /β‐peptides containing β‐amino acids or small β‐peptide fragments as therapeutic peptides.     

3-NITRO-2-PYRIDINESULFENYL (Npys) GROUP

The novel 3-nitro-2-pyridinesulfenyl (Npys) group, which is useful for the protection and the activation of amino and hydroxyl groups for peptide synthesis, is reported. The Npys group is readily introduced by treatment of amino acids with 3-nitro-2-pyridinesulfenyl chloride. The Npys group is easily removed by treatment with very dilute HCl, e.g. 0.1-0.2 N HCl in dioxane, but it is resistant to trifluoroacetic acid and 88% formic acid. Npys is also selectively removed under neutral conditions using triphenylphosphine or 2-pyridinethiol 1-oxide without affecting benzyloxycarbonyl (Z), tert-butyloxycarbonyl (Boc), 2-(4-biphenylyl) propyl(2) oxycarbonyl (Bpoc), 9-fluorenylmethyloxycarbonyl (Fmoc), benzyl (Bzl) or tert-butyl (tBu) protecting groups. The N-Npys and O-Npys groups when activated in the presence of RCOOH by the addition of tertiary phosphine form peptide or ester bonds via oxidation-reduction condensation. The important features of the Npys group are demonstrated through the synthesis of peptides in solution and by solid phase methodology without a formal deprotection procedure. In solid phase synthesis, 4-(Npys-oxymethyl) phenylacetic acid is used as the key intermediate for the introduction of the trifluoroacetic acid resistant 4-(oxymethyl) phenylacetamido linking group to the resin.     

Reductions in beta-amyloid concentrations in vivo by the gamma-secretase inhibitors BMS-289948 and BMS-299897

A primary pathological feature of Alzheimer's disease is beta-amyloid (Abeta)-containing plaques in brain and cerebral vasculature. Reductions in the formation of Abeta peptides by gamma-secretase inhibitors may be a viable therapy for reducing Abeta in Alzheimer's disease. Here we report on the effects of two orally active gamma-secretase inhibitors. BMS-289948 (4-chloro-N-(2,5-difluorophenyl)-N-((1R)-{4-fluoro-2-[3-(1H-imidazol-1-yl)propyl]phenyl}ethyl)benzenesulfonamide hydrochloride) and BMS-299897 (4-[2-((1R)-1-{[(4-chlorophenyl)sulfonyl]-2,5-difluoroanilino}ethyl)-5-fluorophenyl]butanoic acid) markedly reduced both brain and plasma Abeta(1-40) in APP-YAC mice with ED(50) values of 86 and 22 mg/kg per os (po), respectively, for BMS-289948, and 30 and 16 mg/kg po, respectively, for BMS-299897. Both compounds also dose-dependently increased brain concentrations of APP carboxy-terminal fragments, consistent with inhibition of gamma-secretase. BMS-289948 and BMS-299897 (100 mg/kg po) reduced brain and plasma Abeta(1-40) rapidly (within 20min) and maximally within 3 h. BMS-299897 also dose-dependently reduced cortical, cerebrospinal fluid (CSF), and plasma Abeta in guinea pigs with ED(50) values of 30 mg/kg intraperitoneally, without affecting CSF levels of alpha-sAPP. The reductions in cortical Abeta correlated significantly with the reductions in both plasma (r(2) = 0.77) and CSF (r(2) = 0.61) Abeta. The decreases in Abeta were apparent at 3 and 6 h post-administration of BMS-299897, but not at 12h. These results demonstrate that BMS-289948 and BMS-299897 are orally bioavailable, functional gamma-secretase inhibitors with the ability to markedly reduce Abeta peptide concentrations in APP-YAC transgenic mice and in guinea pigs. These compounds may be useful pharmacologically for examining the effects of reductions in beta-amyloid peptides in both animal models and in Alzheimer's disease.     

Synthesis and pharmacological characterization of [I]MRS5127, a high affinity, selective agonist radioligand for the A3 adenosine receptor

A recently reported selective agonist of the human A₃ adenosine receptor (hA₃AR), MRS5127 (1′R,2′R,3′S,4′R,5′S)-4′-[2-chloro-6-(3-iodobenzylamino)-purine]-2′,3′-O-dihydroxy-bicyclo-[3.1.0]hexane, was radioiodinated and characterized pharmacologically. It contains a rigid bicyclic ring system in place of a 5′-truncated ribose moiety, and was selected for radiolabeling due to its nanomolar binding affinity at both human and rat A₃ARs. The radioiodination of the N⁶-3-iodobenzyl substituent by iododestannylation of a 3-(trimethylstannyl)benzyl precursor was achieved in 73% yield, measured after purification by HPLC. [¹²⁵I]MRS5127 bound to the human A₃AR expressed in membranes of stably transfected HEK 293 cells. Specific binding was saturable, competitive, and followed a one-site binding model, with a K_d value of 5.74 ± 0.97 nM. At a concentration equivalent to its K_d, non-specific binding comprised 27 ± 2% of total binding. In kinetic studies, [¹²⁵I]MRS5127 rapidly associated with the hA₃AR (t_1/2 = 0.514 ± 0.014 min), and the affinity calculated from association and dissociation rate constants was 3.50 ± 1.46 nM. The pharmacological profile of ligands in competition experiments with [¹²⁵I]MRS5127 was consistent with the known structure-activity-relationship profile of the hA₃AR. [¹²⁵I]MRS5127 bound with similar high affinity (K_d, nM) to recombinant A₃ARs from mouse (4.90 ± 0.77), rabbit (2.53 ± 0.11), and dog (3.35 ± 0.54). For all of the species tested, MRS5127 exhibited A₃AR agonist activity based on negative coupling to cAMP production. Thus, [¹²⁵I]MRS5127 represents a new species-independent agonist radioligand for the A₃AR. The major advantage of [¹²⁵I]MRS5127 compared with previously used A₃AR radioligands is its high affinity, low degree of non-specific binding, and improved A₃AR selectivity.     

红景天甙的合成

本文改进了以2-(对-氨基苯基)乙醇制备酪醇的操作方法。收率为94.2%,较文献值提高约30%。红景天甙以酪醇为原料,经制得中间体四乙酰基红景天甙后,再脱乙酰基合成而得。本文用中间体的混合物直接脱乙酰基,简化了操作过程。     

Synthesis of higenamine,A cardiotonic principle of aconite root

Higenamine (I), a cardiotonic principle of Aconite root, was synthesized from 4-methoxy-phenylacetic acid (II) and β-(3, 4-dimethoxyphenyl)-ethylamine (IV). Condensation ofIV with 4-methoxyphenylacetyl chloride (III) was followed by cyclodehydration yielding 1-(4′-methoxybenzyl)-6,7-dimethoxy-3,4-dihydroisoquinoline (VI). Reduction ofVI to 1, 2, 3, 4-tetrahydroisoquinoline (VII) and subsequent demethylation provided desired product higenamine, 1-(4′-hydroxybenzyl)-6, 7-dihydroxy-1, 2, 3, 4-tetrahydroisoquinoline.     

Cellular Internalization of Human Calcitonin Derived Peptides in MDCK Monolayers: A Comparative Study with Tat(47-57) and Penetratin(43-58)

PURPOSE: The objective of this study was to evaluate key motif requirements of human calcitonin (hCT)-derived peptides for the permeation through the plasma membrane of MDCK monolayers, as epithelial model. METHODS: Truncated and sequence-modified fluorescent-labeled hCT-derived peptides were synthesized through Fmoc chemistry. Peptide uptake by confluent MDCK was observed by confocal laser scanning microscopy. The cytotoxic effect of the peptides on cellular integrity was followed by LDH release. For direct comparison we covered the cellular uptake of established cell penetrating peptides, Tat(47-57) and penetratin(43-58). RESULTS: Truncated sequences of hCT, from hCT(9-32) to hCT(18-32), penetrated the plasma membrane and demonstrated a sectoral, punctuated cytoplasmic distribution. The uptake process appeared to be temperature-, time- and concentration-dependent. Amino acid modifications of hCT(18-32) indicated that both the proline in position 23 and the positive charge of lysine in position 18 are crucial for peptide uptake. The reverse sequence hCT(32-18) did not penetrate the membrane, indicating the importance of sequence orientation. Tat(47-57) and penetratin(43-58) showed a similar punctuated cytoplasmic distribution in MDCK and HeLa cell lines. No relevant toxicity was observed. CONCLUSIONS: Selected hCT-derived peptides have cell penetrating properties. The uptake mechanism seems to involve an endocytic pathway.     

Airborne polychlorinated biphenyls (PCBs) reduce telomerase activity and shorten telomere length in immortal human skin keratinocytes (HaCat)

PCBs, a group of 209 individual congeners, are ubiquitous environmental pollutants and classified as probable human carcinogens. One major route of exposure is by inhalation of these industrial compounds, possibly daily from inner city air and/or indoor air in contaminated buildings. Hallmarks of aging and carcinogenesis are changes in telomere length and telomerase activity. We hypothesize that semi-volatile PCBs, like those found in inner city air, are capable of disrupting telomerase activity and altering telomere length. To explore this possibility, we exposed human skin keratinocytes to a synthetic Chicago Airborne Mixture (CAM) of PCBs, or the prominent airborne PCB congeners, PCB28 or PCB52 for up to 48 days and determined telomerase activity, telomere length, cell proliferation, and cell cycle distribution. PCBs 28, 52 and CAM significantly reduced telomerase activity from days 18-48. Telomere length was shortened by PCB 52 from day 18 and PCB 28 and CAM from days 30 on. All PCBs decreased cell proliferation from day 18; only PCB 52 produced a small increase of cells in G0/G1 of the cell cycle. This significant inhibition of telomerase activity and reduction of telomere length by PCB congeners suggest a potential mechanism by which these compounds could lead to accelerated aging and cancer.