To elucidate the N-terminal structure–activity relationships of polymyxin B peptides, seven polymyxin B component peptides, the structures of which having been elucidated, and seven N-terminal fatty acid and/or amino acid deletion analogs were synthesized, and their antimicrobial activities determined. The lipopolysaccharide (LPS) binding activities of synthetic peptides were evaluated using [Dab(Dansyl-Gly)1]-polymyxin B3 (Dab; l-α,γ-diaminobutyric acid) as a fluorescent probe. The results indicated that the fatty acyl moiety was not indispensable for LPS binding, but the C9 fatty acyl groups of polymyxin B peptides contributed to the binding affinity to a slightly greater extent than C8 or C7. The fatty acyl moieties of polymyxin B contributed greatly to the antimicrobial activity, while the distinct N-terminal structures of polymyxin B1–B6, bearing normal-, iso-, or anteiso-fatty acids, or 3-hydroxy-fatty acid with chain lengths between C7 and C9, did not affect bactericidal potency.

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We synthesized fifteen oligopeptides consisting of Asp or Glu conjugated with a fluorescent probe, 9- fluorenylmethylchloroformate (Fmoc). In the in vitro binding assay to putative hydroxyapatite (HA), the affinities of these conjugates depended only on the number of amino acid residues, not on their optical characters (L or D) or their species (Asp or Glu). In an in vivo experiment involving a single i.v. injection of Fmoc-D-Asp oligopeptides into mice, peptides consisting of over six Asp residues were selectively distributed to the bone. Then, we synthesized estradiol-17 beta-succinate-(L-Asp)6 [E2-(L-Asp)6] and studied its pharmacokinetic characteristics and its antiosteoporotic effects on ovariectomized (OVX) mice. Although the distribution volume of E2-(L-Asp)6 was significantly smaller than that of E2, E2-(L-Asp)6 was selectively distributed in the bone after i.v. injection and gradually decreased during 7 days. E2-(L-Asp)6 effectively prevented OVX-induced bone loss, without altering the uterine weight, in the dosage range of 0.11 to 1.1 mumol/kg once a week, while E2 increased both the bone mineral density and uterine weight at 0.37 mumol/kg every third day. The results suggest that acidic oligopeptide may be useful for drug delivery to bone and E2-(L-Asp)6 is a good candidate as an anti-osteoporosis drug without the adverse side effects of E2.

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It is now generally documented that the cause of Alzheimer's disease is progressive deposition (histopathologically identified as senile plaque) of the amyloid b protein (Ab) (Ab1-42 and Ab1-40) with aging or due to genetic background in vulnerable brain regions, including the hippocampus and cortex. This deposition is strongly suspected to contribute to disruption of the neuronal network as a result of ubiquitous apoptotic cell death induced by Ab.1,2) The addition of Ab and its hydrophobic core fragments such as Ab25-35 (a monodecapeptide with an amino acid sequence identical to that from 25 to 35 of Ab) has been reported to induce significant cell death in approximately one week in primary cultures of rat hippocampal neurons. [3][4][5] Geniposide and genipin, iridoid compounds found in many plants such as Gardenia jasminoides, have been demonstrated in our laboratory to have neurotrophic, nerve growth factor (NGF)-like activity in their neuritogenesis and signaling promotion in PC12h cells, with genipin having the stronger effect.6,7) Administration of genipin counteracted the trauma-and scopolamine-induced impairment of memory in mice. 8) In some reported experiments, NGF and other neurotrophic factors reversed the deficit in spatial learning and memory in aged rats.9,10) We investigated whether these two iridoids can affect the neuronal toxicity of Ab in cultured hippocampal neurons.Hippocampal neurons from 18-d rat embryos were prepared as described previously.11) Hippocampal cells were maintained for 2 d in 48-well culture plates with Dulbecco's modified Eagle medium (DMEM) supplemented with 10% precolostrum new-born calf serum, selected for neuronal populations by incubation with cytosine-b-D-arabinofuranoside 10 mM for 24 h in Ca 2ϩ , Mg 2ϩ -free phosphate-buffered saline, pH 7.4 (CMF-PBS), and then treated with Ab25-35 (40 mM) in CMF-PBS for 2 d without serum. Ab25-35 was prepared using a peptide synthesizer according to the method described previously.11) It was dissolved in CMF-PBS at a concentration of 1.8 mM (most of the Ab was in fine suspension).Toxic effect on the cultures was evaluated by estimation of lactate dehydrogenase (LDH) release during 2 d of culture in the presence of Ab25-35 with and without test compounds as specified below and according to the method described previously. 12)Geniposide was kindly provided by Toyo F.C.C. Co. (Tokyo, Japan) and genipin was purchased from Wako Pure Chemical Industries (Osaka, Japan). They were dissolved in distilled water and refrigerated until use.The addition of Ab25-35 40 mM to the hippocampal neuron cultures markedly increased LDH release (Fig. 1, Ab25-35 only). Our colleagues previously found in cultured hippocampal cells that Ab25-35-induced plasma membrane impairment was blocked by the addition of the endonuclease inhibitor aurintricarboxylic acid, indicating that the cell degeneration was a result of apoptotic cell death.13) The neurotoxicity of Ab was effectively attenuated by the addition of genipin (20 mM or more) to the medium (Fig. 1A). ...