Enhanced expression of nidogen 1 around the nest of basal cell carcinoma compared with that around squamous cell carcinoma.

Basal cell carcinoma (BCC) is a malignant skin tumor originating from cells of the epidermal basal layer and adnexal epithelium, especially in sun-exposed areas. Unlike squamous cell carcinoma (SCC), BCC has a propensity to grow only locally possibly due to differences in the surrounding microenvironment including the basement membrane (BM) and stroma. To investigate the components constituting the BM and surrounding connective tissue in BCC and SCC, we analyzed the expression of BM proteins, nidogen 1 (NID1) and type IV collagen (COL4). We compared the immunohistochemical expressions of NID1 and COL4 among tumor specimens from BCC, SCC and its precancerous condition, actinic keratosis (AK), (n = 5 each condition). The expressions of NID1 and COL4 were both decreased around the tumor nest of SCC. In contrast, the expressions of both NID1 and COL4 around the nest of BCC were much higher than in the peri-lesional normal skin not only at the BM, but also in the surrounding stromal tissue. Our findings imply that the surrounding stromal cells of BCC, but not SCC or AK, excessively produce NID1 and COL4, which may be involved in preventing BCC cells from destroying the BM and invading the dermis.

Complete right- and left-sided thoracic ducts associated with aberrant left vertebral artery: unreported case with surgical implications.

Anatomy is the keystone to an appropriate understanding of surgical and radiological sciences. Here the authors report on a rare case of complete right- and left-sided thoracic ducts (TDs) associated with aberrant left-vertebral artery (LVA) arising from the aortic arch. The TDs originated from right and left cisterna chyli and terminated separately close to the left venous angle. Superior to the aortic arch, the TDs showed different relationships to the LVA; the right TD was ventral, while the left was dorsal in position. This report is associated with other variations detailed below, and may have important implications in cervicothoracic surgery. (Folia Morphol 2018; 77, 1: 156-160).

Receptor clustering and activation by multivalent interaction through recognition peptides presented on exosomes.

We demonstrate a novel system for inducing clustering of cell surface receptors via recognition peptide segments displayed on exosomes, leading to receptor activation. With this system, targeting of receptor-expressing cells and facilitation of the endocytic uptake of exosomes, which contained the anti-cancer protein saporin, were successfully achieved, leading to cell death.

Peripheral circulation evaluation with near-infrared spectroscopy in skeletal muscle during cardiopulmonary bypass.

BACKGROUND: We designed a non-invasive, observational, real-time study, using near-infrared spectroscopy (NIRS) to assess the in vivo effects of cardiopulmonary bypass (CPB) on patients' skeletal muscle as well as the effects of hemodilution and hypothermia on tissue oxygen delivery during CPB. METHODS: The study included 20 consecutive adult patients undergoing open-heart surgery with CPB. Evaluation parameters for peripheral circulation were measured using the NIRO-200NX and recorded every 30 seconds. To assess how hemodilution influences peripheral circulation parameters, we compared data between a group of patients with hematocrit (Hct) values >22% (high Hct group) and those with Hct values ⩽22% (low Hct group). RESULTS: Changes in the concentration of oxygenated hemoglobin (ΔO2Hb, µmol/L), which flows into the skeletal muscle, was an important factor for deciding the tissue oxygenation index (TOI%), showing the tissue oxygen saturation. The low Hct group showed a significant increase in the normalized tissue hemoglobin index (nTHI), showing the percentage change in the amount of initial hemoglobin and TOI compared to the high Hct group. Changes in the concentration of oxygenated hemoglobin (ΔO2Hb, µmol/L) and deoxygenated hemoglobin (ΔHHb, µmol/L) were significantly less in the low Hct group than in the high Hct group, thus, showing good peripheral circulation despite the low hematocrit levels. CONCLUSION: Our study indicated the presence of a compensatory mechanism in which increased blood flow of the microcirculation is in compensation for the lack of oxyhemoglobin delivery caused by hemodilution.

Arginine-rich peptides and their internalization mechanisms.

As the versatility and use of CPPs (cell-penetrating peptides) as intracellular delivery vectors have been widely accepted, the cellular uptake mechanisms that enable their efficient internalization have become the subject of much interest. Arginine-rich peptides, including HIV-1 Tatp (transactivator of transcription peptide), are regarded as a representative class of CPPs. Evidence suggests that macropinocytosis plays a crucial role in the cellular uptake of these peptides. We have recently shown that treatment of cells with arginine-rich peptides induces activation of Rac protein leading to F-actin (filamentous actin) organization and macropinocytosis. We have also shown that depletion of membrane-associated proteoglycans results in the failure of this signalling pathway, suggesting that membrane-associated proteoglycans may act as a potential receptor for the induction of macropinocytic uptake of arginine-rich peptides. However, when the macropinocytic pathway is inhibited at a low temperature or by cholesterol depletion, these peptides can be internalized by alternative mechanisms, one of which appears to be direct translocation of the peptides through the plasma membrane. This review summarizes the current theories on both endocytic and non-endocytic aspects of internalization of arginine-rich peptides.

Octaarginine-modified multifunctional envelope-type nanoparticles for gene delivery.

This study describes a multifunctional envelope-type nano device (MEND) that mimics an envelope-type virus based on a novel packaging strategy. MEND particles contain a DNA core packaged into a lipid envelope modified with an octaarginine peptide. The peptide mediates internalization via macropinocytosis, which avoids lysosomal degradation. MEND-mediated transfection of a luciferase expression plasmid achieved comparable efficiency to adenovirus-mediated transfection, with lower associated cytotoxicity. Furthermore, topical application of MEND particles containing constitutively active bone morphogenetic protein (BMP) type IA receptor (caBmpr1a) gene had a significant impact on hair growth in vivo. These data demonstrate that MEND is a promising non-viral gene delivery system that may provide superior results to existing non-viral gene delivery technologies.

Activation of c-Jun NH2-terminal kinase (JNK) pathway during islet transplantation and prevention of islet graft loss by intraportal injection of JNK inhibitor.

AIMS/HYPOTHESIS: Although application of the Edmonton protocol has markedly improved the outcome for pancreatic islet transplantation, the insulin independence rate after islet transplantation from one donor pancreas has remained low. During the isolation process and subsequent clinical transplantation, islets are subjected to severe adverse conditions that impair survival and ultimately contribute to graft failure. The aim of this study was to map the c-Jun NH2-terminal kinase (JNK) pathway that mediates islet loss during islet transplantation and to clarify whether intraportal injection with JNK inhibitor during islet transplantation can prevent islet graft loss. METHODS: We measured JNK activity in the liver, fat and muscle of diabetic mice and in the liver immediately after islet transplantation. We examined the effect of intraportal injection of JNK inhibitory peptide at islet transplantation. RESULTS: JNK activity became progressively higher at least until 24 h after transplantation. The cell-permeable peptide of JNK inhibitor was delivered not only in the liver but also in other insulin target organs, preventing JNK activation in the liver at least until 24 h after transplantation and reducing JNK activity in these insulin target organs. Moreover, the peptide inhibitor prevented islet graft loss immediately after transplantation and improved islet transplant outcome. CONCLUSIONS/INTERPRETATION: These findings suggest that control of the JNK pathway is extremely important in islet transplantation and that intraportal injection of JNK inhibitor during islet transplantation (addition of JNK inhibitor to transplant media) could prevent the impairment of islet cells, leading to improved outcome for pancreatic islet transplantation.

The transcriptional landscape of the mammalian genome.

This study describes comprehensive polling of transcription start and termination sites and analysis of previously unidentified full-length complementary DNAs derived from the mouse genome. We identify the 5' and 3' boundaries of 181,047 transcripts with extensive variation in transcripts arising from alternative promoter usage, splicing, and polyadenylation. There are 16,247 new mouse protein-coding transcripts, including 5154 encoding previously unidentified proteins. Genomic mapping of the transcriptome reveals transcriptional forests, with overlapping transcription on both strands, separated by deserts in which few transcripts are observed. The data provide a comprehensive platform for the comparative analysis of mammalian transcriptional regulation in differentiation and development.

Quantitative three-dimensional analysis of the intracellular trafficking of plasmid DNA transfected by a nonviral gene delivery system using confocal laser scanning microscopy.

Since endosomal escape and the nuclear delivery of plasmid DNA (pDNA) constitute major barriers for transgene expression, a quantitative evaluation of intracellular trafficking of pDNA would be highly desirable in terms of optimizing a nonviral gene delivery system. In the present study, a novel strategy is proposed for the quantification of rhodamine-labeled pDNA in endosomes/lysosomes, cytosol, and nucleus. Endosomes/lysosomes and nucleus were stained with LysoSensor DND-189 and Hoechst 33258, respectively, to distinguish them from the cytosol. The pixel areas of the clusters derived from the rhodamine were used as an index for the amount of pDNA. This approach was applied to the analysis of the intracellular trafficking of pDNA transfected by LipofectAMINE PLUS, stearylated octaarginine (STR-R8), and octaarginine (R8). In the case of R8, most of the pDNA was trapped by endosomes/lysosomes. STR-R8 exhibited endosomal escape followed by nuclear translocation in a time-dependent manner. LipofectAMINE PLUS was the most effective in rapidly delivering pDNA to the nucleus as well as the cytosol. These differences in the intracellular trafficking of pDNA correlated well with the transgene expression. Therefore, this method enables the quantitative analysis of the intracellular pharmacokinetics of pDNA and promises to provide useful information for optimizing nonviral gene delivery systems.

Mechanism of improved gene transfer by the N-terminal stearylation of octaarginine: enhanced cellular association by hydrophobic core formation.

The internalization mechanisms associated with octaarginine and stearyl-octaarginine were investigated using confocal laser microscopy and flow cytometric analysis. Octaarginine is able to translocate through cell membranes in a manner that does not exactly involve the classical endocytic pathways of internalization. However, when a stearyl moiety is attached to the N-terminus of octaarginine, the internalization shifts mainly to an endocytosis-dependent pathway. The transfection efficiency of stearyl-octaarginine was significantly higher than that of octaarginin. To understand the mechanism of the improved gene transfer by the N-terminal stearylation of octaarginine, the gene transfer processes mediated by octaarginine or stearyl-octaarginine were compared. Both octaarginine and stearyl-octaarginine are able to carry plasmid DNA into cells. The amount of plasmid DNA internalized as well as that delivered to the nucleus was higher in the case of stearyl-octaarginine. Even though the internalization mechanisms of octaarginine and stearyl-octaarginine were different, their complexes with plasmid DNA were internalized via the same pathway, presumably, the clathrin-mediated pathway of endocytosis. The results of the atomic force microscopy revealed that stearyl-octaarginine, but not octaarginine, can completely condense the DNA into stable complexes that can be highly adsorbed to the cell surface and subsequently highly internalized. Therefore, using stearylated-octaarginine provided higher internalization of plasmid DNA into cells, due to enhanced cellular association, as well as higher nuclear delivery. The results presented in this study provide a better understanding of the mechanisms of improved transfection using stearylated-octaarginine. The concept of using stearylated peptides may aid in the development of more efficient nonviral gene vectors.

Relationship of the fistulas to the rectum and genitourinary tract in mouse fetuses with high anorectal malformations induced by all-trans retinoic acid.

Since high anorectal malformations with fistulae in human embryos and fetuses of successive developmental stages have not been reported, the embryologic relationship between the rectal fistula (RF) and the genitourinary tract (GUT) in high anorectal agenesis (ARA) remains to be elucidated. This study investigates the developmental relationship between the RF and the GUT in male and female fetuses with high ARA using our established model for high ARA with fistula in mice. Pregnant mice received all-trans retinoic acid suspended in corn oil (5 mg/ml) 100 mg/kg i.p. on day 9 of pregnancy. All fetuses were removed from the uterus on a single day from days 12 to 18 of pregnancy. The caudal regions were analyzed histologically with hematoxylin and eosin staining. All fetuses examined had high ARA with fistula. On day 12 of pregnancy, an anomalous communication was seen between the urogenital sinus (UGS) and the rectum. In the affected female fetuses, on day 14 of pregnancy the paramesonephric (müllerian) ducts and müllerian tubercle were located above the rectocloacal fistula (RCF), and on day 18 of pregnancy the uterovaginal canal was located between the cloaca and the RCF. In the male fetuses, on day 14 of pregnancy the junction between the mesonephric (wolffian) duct and the UGS was located away from the junction between the rectum and the UGS. On day 18 of pregnancy the ejaculatory duct was located between the urinary bladder and the rectourethral fistula. The results of our experiment clearly show the embryologic relationship between the RF and the GUT with high ARA. The anomalous communication between the UGS and the rectum may interfere with normal caudal migration along the dorsal wall of the UGS at the junction between the UGS and the mesonephric or paramesonephric duct.

Alamethicin-leucine zipper hybrid peptide: a prototype for the design of artificial receptors and ion channels.

In this report, we describe a novel concept of extramembrane control of channel peptide assembly and the eventual channel current modulation. Alamethicin is a peptide antibiotic, which usually forms ion channels in various association states. By introducing an extramembrane leucine zipper segment (Alm-LeuZ), the association number of alamethicin was effectively controlled to produce a single predominant channel open state. The assembly was estimated to be a tetramer, by comparison of the channel conductance with that of the template-assembled Alm-LeuZ tetramer, which was prepared by the conjugation of a maleimide-functionalized peptide template with cysteine-derivatized Alm-LeuZ segments. Employment of an extramembrane segment of a random conformation provided higher levels of channel conductance. The result exemplified the possibility of channel current control by a conformational switch of the extramembrane segments.

Stearylated arginine-rich peptides: a new class of transfection systems.

Membrane-permeable arginine-rich peptides, such as HIV-1 Tat-(48-60), HIV-1 Rev-(34-50), and flock house virus (FHV) coat-(35-49), have been shown to possess the ability to transfect COS-7 cells with luciferase-coding plasmid as efficiently as polyarginine (MW 5000-15 000) and polylysine (MW 9800). Not only these virus-derived cationic peptides but also oligoarginines of 4-16 residues were found to be able to transfect cells. In the case of the Tat, FHV, and octaarginine peptides, N-terminal stearylation of the peptides increases the transfection efficiency by approximately 100 times to reach the same order of magnitude as that of LipofectAMINE, one of the most efficient commercially available transfection agents. Also, a certain correlation was observed between the transfection efficiency of stearyl-(Arg)n peptides (stearyl-Rn: n = 4, 8, 12, 16) and the membrane permeability of the corresponding (Arg)n peptides (Rn).

Specific cytotoxicity of antibody to YAL-198, a sperm antigen peptide, to murine zygote.

Active immunization with the peptide segments rSMP-230 and YAL-198, corresponding to the hydrophilic extracellular domain of two human sperm antigens (rSMP-B and YWK-II, respectively), reduced fertility in female rats by different mechanisms. The anti-rSMP-230 antibody interferes with human and murine fertilization, and the anti-YAL-198 antibody blocks the development of mouse embryos. The authors examined in vitro at which stage the antibodies to rSMP-230 and YAL-198 were cytotoxic to murine embryos up to morula/blastocyst stage. Anti-rSMP-230 antibody was not cytotoxic to any stages. On the other hand, the anti-YAL-198 antibody arrested the growth of embryos at the 2-cell stage but not at more advanced developmental stages. When the anti-YAL-198 antibody was used, spotty staining was observed only on the surfaces of embryos that had arrested at the 2-cell stage. Unstained embryos, however, continued to develop normally. In contrast, the anti-rSMP-230 antibody stained murine sperm but failed to stain murine ova and embryos. The present results suggest that the human sperm components rSMP-B and YWK-II play important roles in sperm-egg interaction and early development of the embryo, respectively.

Facile solid-phase synthesis of sulfated tyrosine-containing peptides: Part II. Total synthesis of human big gastrin-II and its C-terminal glycine-extended peptide (G34-Gly sulfate) by the solid-phase segment condensation approach.

Application of the fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase segment condensation approach to the preparation of sulfated peptides was investigated through the synthesis of human big gastrin-II, a 34-residue sulfated tyrosine [Tyr(SO3H)]-containing peptide. Highly acid-sensitive 2-chlorotrityl resin (Clt resin) was exclusively employed as an anchor-resin for the preparation of the three peptide segments having the C-terminal Pro residue as well as of the Tyr(SO3H)-containing resin-bound segment. By using the PyBOP-mediated coupling protocol [PyBOP=benzotriazolyloxytris(pyrrolidino)phosphonium hexafluorophosphatel, we successively condensed each segment and constructed the 34-residue peptide-resin without any difficulty. The final acid treatment of the fully protected peptide-resin at low temperature (90% aqueous TFA, 0 degree C for 8 h), which can detach a Tyr(SO3H)-containing peptide from the resin and remove the protecting groups concurrently with minimum deterioration of the sulfate, afforded a crude sulfated peptide. After one-step HPLC purification, a highly homogeneous human big gastrin-II was easily obtained in 14% yield from the protected peptide-resin. The sulfate form of the C-terminal glycine-extended gastrin (G34-Gly sulfate), a posttranslational processing intermediate of gastrin-II, was also successfully prepared with the segment condensation approach (11% yield). These results demonstrated the usefulness of the segment condensation protocol for preparing large Tyr(SO3H)-containing peptides.

Facile solid-phase synthesis of sulfated tyrosine-containing peptides: total synthesis of human big gastrin-II and cholecystokinin (CCK)-39.

Chemical synthesis of tyrosine O-sulfated peptides is still a laborious task for peptide chemists because of the intrinsic acid-lability of the sulfate moiety. An efficient cleavage/deprotection procedure without loss of the sulfate is the critical difficulty remaining to be solved for fluoren-9-ylmethoxycarbonyl (Fmoc)-based solid-phase synthesis of sulfated peptides. To overcome the difficulty, TFA-mediated solvolysis rates of a tyrosine O-sulfate [Tyr(SO3H)] residue and two protecting groups, tBu for the hydroxyl group of Ser and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) for the guanidino group of Arg, were examined in detail. The desulfation obeyed first-order kinetics with a large entropy (59.6 J.K-1.mol-1) and enthalpy (110.5 kJ.mol-1) of activation. These values substantiated that the desulfation rate of the rigidly solvated Tyr(SO3H) residue was strongly temperature-dependent. By contrast, the SN1-type deprotections were less temperature-dependent and proceeded smoothly in TFA of a high ionizing power. Based on the large rate difference between the desulfation and the SN1-type deprotections in cold TFA, an efficient deprotection protocol for the sulfated peptides was developed. Our synthetic strategy for Tyr(SO3H)-containing peptides with this effective deprotection protocol is as follows: (i) a sulfated peptide chain is directly constructed on 2-chlorotrityl resin with Fmoc-based solid-phase chemistry using Fmoc-Tyr(SO3Na)-OH as a building block; (ii) the protected peptide-resin is treated with 90% aqueous TFA at 0 degree C for an appropriate period of time for the cleavage and deprotection. Human cholecystokinin (CCK)-12, mini gastrin-II (14 residues), and little gastrin-II (17 residues) were synthesized with this method in 26-38% yields without any difficulties. This method was further applied to the stepwise synthesis of human big gastrin-II (34 residues), CCK-33 and -39. Despite the prolonged acid treatment (15-18 h at 0 degree C), the ratios of the desulfated peptides were less than 15%, and the pure sulfated peptides were obtained in around 10% yields.

Evaluation of two sperm antigens, rSMP-B and YWK-II, as targets for immunocontraception.

To determine whether sperm membrane components, rSMP-B and YWK-II, are suitable candidates as immunocontraceptives in humans, antifertility activities of the antibodies to the peptide fragments, rSMP-229 and rSMP-230 of rSMP-B and YAL-198 of YWK-II, were examined. In a previous report, anti-rSMP-230 antibody was shown to immobilise human sperm and to block human fertilisation, and the antigen (rSMP-230) to interact with antisperm antibodies found in sera of infertile women. Antibody to the second synthetic peptide, rSMP-229, corresponding to a different segment of rSMP-B, mimicked the biological activities of the anti-rSMP-230 antibody. Anti-YAL-198 antibody significantly, although weakly, inhibited human fertilisation. In the murine model, the anti-rSMP-B antibodies blocked in vitro fertilisation of mouse eggs but had no influence on embryo growth. Anti-YAL-198 antibody, however, arrested the growth of zygotes. In conclusion, rSMP-B, a human sperm protein, is a promising candidate in the development of an immunocontraceptive for human application. A second sperm protein, YWK-II, is effective as an antifertility immunogen in experimental animals.

A 'cassette' RNase: site-selective cleavage of RNA by RNase S equipped with RNA-recognition segment.

RNase S is a unique protein comprising the non-covalent association of two components, the S-peptide and the S-protein. An RNA-recognition segment derived from the human immunodeficiency virus (HIV)-1 Rev protein was conjugated with the S-peptide to form a complex with the S-protein. The resulting RNase S bearing the RNA-recognition segment preferentially hydrolyzed a single position of the RNA stem-loop derived from the specific binding site for the Rev protein.

Stoichiometry of subunit e in rat liver mitochondrial H(+)-ATP synthase and membrane topology of its putative Ca(2+)-dependent regulatory region.

Previous studies have revealed that residues 34-65 of subunit e of mitochondrial H(+)-ATP synthase are homologous with the Ca(2+)-dependent tropomysin-binding region for troponin T and have suggested that subunit e could be involved in the Ca(2+)-dependent regulation of H(+)-ATP synthase activity. In this study, we determined the content of subunit e in H(+)-ATP synthase purified from rat liver mitochondria, and we also investigated the membrane topology of a putative Ca(2+)-dependent regulatory region of subunit e using an antibody against peptide corresponding to residues 34-65 of subunit e. Quantitative immunoblot analysis of subunit e in the purified H(+)-ATP synthase revealed that 1 mol of H(+)-ATP synthase contained 2 mol of subunit e. The ATPase activity of mitoplasts, in which the C-side of F(0) is present on the outer surface of the inner membrane, was significantly stimulated by the addition of the antibody, while the ATPase activity of submitochondrial particles and purified H(+)-ATP synthase was not stimulated. The antibody bound to mitoplasts but not to submitochondrial particles. These results suggest that the putative Ca(2+)-dependent regulatory region of subunit e is exposed on the surface of the C-side of F(0) and that subunit e is involved in the regulation of mitochondrial H(+)-ATP synthase activity probably via its putative Ca(2+)-dependent regulatory region.

Arginine-rich peptides. An abundant source of membrane-permeable peptides having potential as carriers for intracellular protein delivery.

A basic peptide derived from human immunodeficiency virus (HIV)-1 Tat protein (positions 48-60) has been reported to have the ability to translocate through the cell membranes and accumulate in the nucleus, the characteristics of which are utilized for the delivery of exogenous proteins into cells. Based on the fluorescence microscopic observations of mouse macrophage RAW264.7 cells, we found that various arginine-rich peptides have a translocation activity very similar to Tat-(48-60). These included such peptides as the d-amino acid- and arginine-substituted Tat-(48-60), the RNA-binding peptides derived from virus proteins, such as HIV-1 Rev, and flock house virus coat proteins, and the DNA binding segments of leucine zipper proteins, such as cancer-related proteins c-Fos and c-Jun, and the yeast transcription factor GCN4. These segments have no specific primary and secondary structures in common except that they have several arginine residues in the sequences. Moreover, these peptides were able to be internalized even at 4 degrees C. These results strongly suggested the possible existence of a common internalization mechanism ubiquitous to arginine-rich peptides, which is not explained by a typical endocytosis. Using (Arg)(n) (n = 4-16) peptides, we also demonstrated that there would be an optimal number of arginine residues (n approximately 8) for the efficient translocation.