Cysteinylprolyl ester-mediated drug release from a lipid-drug conjugate.

For small-molecule drugs, lipidation via a cleavable linkage can extend half-life in circulation through interaction with albumin. Here we modified the cysteinylprolyl ester (CPE) system used in peptide thioester synthesis, which normally requires basic conditions, for use as an self-immolative linker and release device for a lipid-gemcitabine conjugate. To improve release under physiological conditions for medical application, a methyl group at the α-position of cysteine on the CPE unit was incorporated in anticipation of the Thorpe-Ingold effect. As a result, Ac-Gly-(α-Me)Cys(SH)-Pro-gemcitabine 11 drastically promoted the release of gemcitabine in comparison with Ac-Gly-Cys(SH)-Pro-gemcitabine 10. Furthermore, in the presence of bovine serum albumin and/or 2-mercaptoethanesulfonic acid, the gentle and continuous release of gemcitabine from the lipid-gemcitabine conjugate 16 was achieved. In addition to gemcitabine, this method could allow high clearance drugs, including nucleic acid and prostacyclin derivatives, to maintain their biological activity long enough to become effective.

Late-Stage Formation of a Sactionine Linkage Enabled by Lossen Rearrangement of Glycyl Hydroxamic Acid.

Late-stage formation of a sactionine thioether bond connecting a Gly α-carbon and Cys thiol was achieved by Lossen rearrangement of a glycyl hydroxamic acid (GlyHA) residue in a peptide. Lossen rearrangement allowed conversion of GlyHA within a peptide to an N-acyl iminium equivalent, which subsequently reacted with S-acetamidomethyl Cys (Cys(Acm)) in TFA in the presence of guanidine hydrochloride (Gn·HCl) to yield the desired thioether linkage in the final stage.

Synthesis of N-Glyoxylyl Peptides Enabled by a Lossen Rearrangement-Induced Intramolecular Redox Reaction of N-Terminal Glycyl Hydroxamic Acid.

An oxidant-free approach to the synthesis of N-glyoxylyl peptides has been developed that utilizes the Lossen rearrangement of the N-terminal glycyl hydroxamic acid residue. The synthesis proceeds via an intramolecular redox mechanism to yield the glyoxylyl peptides, which are then subjected to various peptide cyclization procedures. The reaction scheme is suitable for oxidation-sensitive moieties including amino acids.

Advanced Insulin Synthesis by One-pot/Stepwise Disulfide Bond Formation Enabled by S-Protected Cysteine Sulfoxide.

An advanced insulin synthesis is presented that utilizes one-pot/stepwise disulfide bond formation enabled by acid-activated S-protected cysteine sulfoxides in the presence of chloride anion. S-chlorocysteine generated from cysteine sulfoxides reacts with an S-protected cysteine to afford S-sulfenylsulfonium cation, which then furnishes the disulfide or reversely returns to the starting materials depending on the S-protection employed and the reaction conditions. Use of S-acetamidomethyl cysteine (Cys(Acm)) and its sulfoxide (Cys(Acm)(O)) selectively give the disulfide under weak acid conditions in the presence of MgCl2 even if S-p-methoxybenzyl cysteine (Cys(MBzl)) and its sulfoxide (Cys(MBzl)(O)) are also present. In contrast, the S-MBzl pair yields the disulfide under more acidic conditions in the presence of a chloride anion source. These reaction conditions allowed a one-pot insulin synthesis. Additionally, lipidated insulin was prepared by a one-pot disulfide-bonding/lipidation sequence.

S-Protected Cysteine Sulfoxide-Enabled Tryptophan-Selective Modification with Application to Peptide Lipidation.

Lipidation of peptides is a promising means of modification that can improve the therapeutic character of biologically active peptides. Here, a novel lipidation protocol for peptides is described. The C-H sulfenylation of indole in peptides using S-p-methoxybenzyl cysteine sulfoxide under acidic conditions in the presence of ammonium chloride, anisole, and triisopropylsilane enables late-stage tryptophan-selective peptide lipidation. This developed protocol has been used successfully for the lipidation of glucagon-like peptides. Oral glucose tolerance tests in wild-type mice indicated that the resulting lipidated peptides stimulate insulin secretion and exhibit a more long-lasting blood-glucose-lowering effect than a parent nonlipidated peptide.

Copper(II)-mediated C-H sulphenylation or selenylation of tryptophan enabling macrocyclization of peptides.

Cu(II)-mediated C-H sulphenylation or selenylation of Trp indole by a derivative of cysteine or selenocysteine enables access to the tryptathionine unit or its selenium congener. The mechanism of these protocols, which allow macrocyclization of Trp-containing peptides, has been studied.

Simultaneous dispersion and dispersion-slope compensator based on a doubly sampled ultrahigh-channel-count fiber Bragg grating.

A simultaneous dispersion and dispersion-slope compensator based on a doubly sampled fiber Bragg grating (FBG) with 153 channels has been demonstrated, which is obtained by simultaneously chirping the period of the seed grating and the periods of the two sampling functions. These two sampling functions are optimized with the simulated annealing algorithm and the Gerchberg-Saxton iterative method. Moreover, the spectral inclination (in reflection) inherently due to the different magnitudes of the channel dispersion has been successfully equalized.

Advanced design of the ultrahigh-channel-count fiber Bragg grating based on the double sampling method.

A double sampling method enabling to have excellent channel uniformity and high in-band energy efficiency is firstly proposed for the design of an ultrahigh-channel-count fiber Bragg grating (FBG), which is based on the simultaneously utilization of an amplitude-assisted phase sampling (AAPS) function and a phase-only sampling (POS) function. As examples, two typical 10-dB FBGs with a length of 12 cm, dispersion of - 1360 ps/nm, channel spacing of 0.8 nm, and a consecutive 135- and 405- channels are numerically designed. The maximum index-modulations required are about 0.8x10(-3), and 1.3x10(-3), respectively. Compared with the proposed method, the other two kinds of double sampling schemes by utilizing either the double AAPS (i.e., AAPS+AAPS) or the double POS (i.e., POS+POS) have also been introduced for the design of the multichannel FBGs. Fabrication tolerances to the designed 135-channel FBG obtained with the AAPS plus POS method are numerically investigated.

Ultrahigh-channel-count phase-only sampled fiber Bragg grating covering the S, C, and L bands.

A continuous phase-only sampling scheme enabling one to create an ultrahigh-channel-count fiber Bragg grating (FBG) with excellent channel uniformity and high in-band energy efficiency is proposed that is based on the simultaneous utilization of two phase-only sampling functions. With this method, a linearly chirped 135-channel FBG with a length of 10 cm, a dispersion of 1360 ps/nm, channel spacing of 0.8 nm, and grating strength of 10 dB is theoretically demonstrated, and the maximum refractive index change required is less than 10(-3).

Neuronal expression of Fas-associated death domain protein and caspase-8 in the perinidal parenchyma of cerebral arteriovenous malformations.

OBJECT: The expression and localization of phosphorylated Fas-associated death domain protein (pFADD) and cleaved caspase-8 was examined in human cerebral arteriovenous malformations (AVMs). The authors focused on the perinidal parenchyma to clarify the effect of AVMs on perinidal brain tissue. METHODS: Seventeen cerebral AVMs were analyzed using immunohistochemical methods. Specimens were removed from patients during surgical procedures. The characteristics of the areas that stained positively for pFADD or cleaved caspase-8 were also assessed using an image analysis system. Eleven (65%) of the 17 lesions demonstrated anti-pFADD immunoreactivity and 12 (71%) showed anti-cleaved caspase-8 immunoreactivity. The immunoreactive cells in the perinidal parenchyma demonstrated obvious neuronal morphological characteristics. The characteristics of pFADD-positive and cleaved caspase-8-positive areas were assessed using the image analysis system. The mean distance from the nidus adjacent to either area was not affected by preoperative hemorrhage. The neuronal densities of pFADD-positive and cleaved caspase-8-positive areas were analyzed using the same system. The density of the control area (samples that were pFADD-negative and cleaved caspase-8 negative) was significantly higher when compared with that of pFADD-positive and cleaved caspase-8-positive areas (p < 0.05). The expressions of cleaved caspase-9, cleaved poly(adenosine diphosphate-ribose) polymerase, and apoptotic cells were analyzed using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method. CONCLUSIONS: Neuronal areas that stained positively for pFADD and cleaved caspase-8 existed around the nidus of AVMs. In these areas, the neuronal density was lower than that in the other parenchyma around the AVM. Neuronal loss around the nidus may be the origin of brain dysfunction around AVMs.

Expression of hypoxia-inducing factor-1 alpha and endoglin in intimal hyperplasia of the middle cerebral artery of patients with Moyamoya disease.

OBJECTIVE: Moyamoya disease (MMD) is a cerebrovascular occlusive disease characterized by progressive stenosis or occlusion at the distal ends of the bilateral internal arteries. In MMD, intimal hyperplasia was previously reported to be found in autopsy samples. In this study focusing on the mechanism of remodeling of the intracranial arterial walls of patients with MMD, we surgically collected tiny pieces of the wall of the middle cerebral artery (MCA) from patients with MMD and analyzed them using histological and immunohistochemical methods. METHODS: Twelve patients underwent surgical procedures for treatment of standard indications of MMD at Kyoto University Hospital. Specimens of MCA were obtained from MMD patients during the surgical procedures. Nine MCA samples were also obtained in the same way from control patients. The samples were analyzed by immunohistochemical methods. RESULTS: MCA specimens from MMD patients had a thicker intima than those from the control group. In MMD samples, the immunoreactivity indicating hypoxia-inducing factor-1alpha was higher in the endothelium and intima; endoglin expression was also higher in the endothelium. No vascular endothelial growth factor immunoreactivity was detectable in the MMD samples. In addition, transforming growth factor-beta3 immunoreactivity was also detected and was co-localized with that of hypoxia-inducing factor-1alpha and endoglin, mainly in the endothelium. CONCLUSION: Our results indicate that the MCA specimens from MMD patients had thicker intimal walls than the specimens from control patients. In addition, hypoxia-inducing factor-1alpha and endoglin were overexpressed in the intima of the MCA of MMD patients.

Internal carotid artery obstruction derived from persistent arterial wall dissection associated with old trivial trauma.

A 59-year-old man presented with acute onset of visual loss in his right eye. He was treated under a diagnosis of retinal artery thrombosis. Ultrasonography revealed obstruction of the ipsilateral internal carotid artery (ICA). He had no risk factor for stroke and he denied any history of trauma. Follow-up ultrasonography obtained 6 months later showed spontaneous ICA recanalization. Cerebral angiography demonstrated an arterial wall flap suggesting ICA dissection at the craniocervical junction. He then remembered suffering hemicranial headache and Horner's sign of several days' duration after jumping off a stepladder 1 year earlier. The present case is quite unusual in that persistent carotid arterial wall dissection was thought to proceed to ICA obstruction and manifested as retinal ischemia after a long asymptomatic period.

Primate embryonic stem cell-derived neuronal progenitors transplanted into ischemic brain.

Transplantation of stem cells has the possibility of restoring neural functions after stroke damage. Therefore, we transplanted neuronal progenitors generated from monkey embryonic stem (ES) cells into the ischemic mouse brain to test this possibility. Monkey ES cells were caused to differentiate into neuronal progenitors by the stromal cell-derived inducing activity method. Focal cerebral ischemia was induced by occluding the middle cerebral artery by the intraluminal filament technique. The donor cells were transplanted into the ischemic lateral striatum at 24 h after the start of reperfusion. The cells transplanted into the ischemic brain became located widely around the ischemic area, and, moreover, the transplanted cells differentiated into various types of neurons and glial cells. Furthermore, at 28 days after the transplantation, over 10 times more cells in the graft were labeled with Fluorogold (FG) by stereotactic focal injection of FG into the anterior thalamus and substantia nigra on the grafted side when compared with the number at 14 days. From these results we confirmed the survival and differentiation of, as well as network formation by, monkey ES-cell-derived neuronal progenitors transplanted into the ischemic mouse brain.

Survival and differentiation of neural progenitor cells derived from embryonic stem cells and transplanted into ischemic brain.

OBJECT: Cell replacement therapy including the use of embryonic stem cells (ESCs) may represent a novel treatment for damage from stroke. In this study, the authors transplanted neural progenitor cells (NPCs) derived from ESCs into ischemic brain and analyzed their survival and differentiation. METHODS: Multipotential NPCs were generated from ESCs by using the stromal cell-derived inducing activity method. These cells could differentiate in vitro into neurons, glia, and oligodendrocytes, thus revealing them to be neural stem cells. The NPCs were then transplanted into ischemic brain. At 2 weeks postischemia, the transplanted cells occupied 18.8 +/-2.5% of the hemispheric area; by 4 weeks postischemia, 26.5 +/- 4% of the hemisphere. At 4 weeks after transplantation, green fluorescent protein (GFP)-positive transplanted cells showed mature neuronal morphological features. The authors also investigated the expression of differentiation markers and various neurotransmitters. Transplanted cells were immunopositive for neuronal nuclei, beta-tubulin-III, and glial fibrillary acidic protein. Of the GFP-positive cells, 33.3 +/-11.5% were positive for glutamate decarboxylase, 13.3 +/- 5.8% for glutamate, 2.1 +/- 2.5% for tyrosine hydroxylase, 1.8 +/- 2% for serotonin, and 0.4 +/- 0.2% for choline acetyltransferase. CONCLUSIONS: The authors confirmed the survival and differentiation of ESC-derived NPCs transplanted into the ischemic brain. Surviving transplanted cells expressed several neural markers and neurotransmitters. These findings indicate that these cells can function in the brain.

Activation of p38 kinase in the gerbil hippocampus showing ischemic tolerance.

Ischemic tolerance is a phenomenon in which brief episodes of ischemia protect against the lethal effects of subsequent periods of prolonged ischemia. The authors investigated the activation of p38 mitogen-activated protein kinase (p38) in the gerbil hippocampus by Western blotting and immunohistochemistry to clarify the role of p38 kinase in ischemic tolerance. After the 2-minute global ischemia, immunoreactivity indicating active p38 was enhanced at 6 hours of reperfusion and continuously demonstrated 72 hours after ischemia in CA1 and CA3 neurons. Pretreatment with SB203580, an inhibitor of active p38 (0-30 micromol/l), 30 minutes before the 2-minute ischemia reduced the ischemic tolerance effect in a dose-dependent manner. Immunoblot analysis indicated that alteration of the phosphorylation pattern of p38 kinase in the hippocampus after subsequent lethal ischemia was induced by the preconditioning. These findings suggest that lasting activation of p38 may contribute to ischemic tolerance in CA1 neurons of the hippocampus and that components of the p38 cascade can be target molecules to modify neuronal survival after ischemia.

A new method to prepare multicellular spheroids in cancer cell lines using a thermo-reversible gelation polymer.

The purpose of this study is to utilize the thermo-reversible gelation polymer in which the sol-gel transitting phase is reversibly changed by temperature in a three-dimensional culture system. Human cancer cells have been observed to form multicellular spheroids, whereas fibroblasts slowly develop into small spheroids with the culture medium including this polymer. This polymer has some advantages for use as a culture material, as follows: first, cancer cells grow three-dimensionally in the aqueous solution of this polymer; second, it is easy to harvest cells or spheroids in the aqueous solution of this polymer by simply cooling down the temperature; and third, the culture medium including this polymer is so translucent that the cells or spheroids can be observed through a phase-contrast microscope. We thus conclude that this polymer is a very useful material for three-dimensional cultures.

Adenovirus-mediated gene transfer of fibroblast growth factor-2 increases BrdU-positive cells after forebrain ischemia in gerbils.

BACKGROUND AND PURPOSE: Progenitor cells continue to generate neurons in the adult mammalian brain, and cerebral ischemia induces neurogenesis. We examined the efficacy of the intraventricular injection of a recombinant adenovirus-expressing fibroblast growth factor-2 (FGF-2) (AxCAMAssbFGF) on neurogenesis in both normal and ischemic brains. METHODS: We used a gerbil model of transient global ischemia and counted the number of BrdU-positive cells after injection of AxCAMAssbFGF into the brain with or without ischemia. RESULTS: Intraventricular AxCAMAssbFGF produced robust FGF-2 protein increases in diverse regions of the brain and markedly increased FGF-2 concentrations in cerebrospinal fluid 2 days after administration and evoked significant proliferation of BrdU-positive cells not only in the subventricular zone and dentate gyrus of the hippocampus but also in the cerebral cortex, and some BrdU-positive cells differentiated into neurons. Continuous intraventricular infusion of FGF-2 protein increased FGF-2 concentration in cerebrospinal fluid but not in brain tissues and produced BrdU-positive cell proliferation only in the subventricular zone of the lateral ventricle. CONCLUSIONS: Adenovirally mediated transfer of the FGF-2 gene promoted progenitor cell proliferation more efficiently in widespread regions of the brain after transient global ischemia than continuous intraventricular infusion of FGF-2 protein.