Conformational dependence of integrin-binding peptides derived from homologous loop regions in the laminin α chains.

Laminin α chains (α1-α5 chains) are expressed in a tissue- and developmental stage-specific manner and have diverse chain-specific biological functions. Especially, laminin globular (LG) modules (LG1-LG5) located at the C-terminus of the α chains play a critical role in the biological activities of laminins. Each LG module is composed of a 14-stranded ß-sheet (A-N) sandwich structure. We previously screened cell attachment activity of the loop regions between the E and F strands in the LG modules using 17 homologous peptides (EF peptides) and found that four active EF peptides bind to integrin α2ß1. One of the four peptides, G4EF1 demonstrated improved cell attachment activity when cyclized. Here, we focused on the remaining three integrin α2ß1-binding EF peptides (G5EF1, G3EF3, and G5EF5) and analyzed the relationship between their peptide conformation and cell attachment activity. First, we determined their active core sequences and found that G5EF1z (IGLEIVDGKVLFHVNN), G3EF3z (LLVTLEDGHIALST), and G5EF5z (KVLTEQVL) are the core sequences. Cyclic peptides of the core sequences (cycloG5EF1z, cycloG3EF3z, and cycloG5EF5z) enhanced integrin-mediated cell adhesion activity compared with their linear peptides. The results indicated that cell adhesion activity of the integrin α2ß1-binding EF peptides is conformation dependent and that the loop structure is critical for their activity. This suggests that conformation of the loop regions plays an important role for the activities of the LG modules.

Identification of active sequences in human laminin α5 G domain.

Human laminin-511 (α5ß1γ1) and its truncated protein, laminin-511 E8 fragment, bind to integrin α6ß1 and have been widely used for embryonic stem cell and induced pluripotent stem cell culture under feeder-free conditions. In this study, we focused on human laminin α5 chain G domain, which is thought to be critical for the biological functions of laminin-511, and screened its biologically active sequences using a synthetic peptide library. We synthesized 115 peptides (hA5G1-hA5G115) covering the entire laminin α5 chain G domain and evaluated cell attachment activity using both the peptide-coated plate and peptide-chitosan matrix (peptide-ChtM) assays. Seventeen peptides demonstrated cell attachment activity in the assays. Both hA5G18 and hA5G26-coated plates and hA5G74-ChtMs promoted integrin ß1-mediated cell attachment. These findings are useful for the study of molecular mechanisms of laminin-511, and the active peptides have a potential for use as a molecular probe for cell adhesion receptors.

Prediction of clinical effects of infliximab administered for inflammatory bowel disease based on pharmacokinetic and pharmacodynamic modeling.

Infliximab (IFX) is used as a therapeutic agent for ulcerative colitis (UC) and Crohn's disease (CD). Although the dosage regimen has been established through clinical trial experience, it has yet to be assessed with a pharmacokinetic and pharmacodynamic model. The present study analysed sequential changes of clinical response in patients with ulcerative colitis and Crohn's disease following repeated administrations of infliximab using the pharmacokinetic/pharmacodynamic model. In addition, the dosage regimen presently used for patients with ulcerative colitis was evaluated, as well as the potential efficacy gained by increasing the dose and/or reducing the interval of administration for patients with Crohn's disease. Furthermore, the possibility of evaluating the difference between both diseases with regard to the efficacy of infliximab was investigated. Sequential changes in the clinical response values obtained with our model were in good agreement with the observed values following administration of infliximab in patients with ulcerative colitis and Crohn's disease. The results showed the importance of a loading dose for patients with ulcerative colitis, as well as the efficacy of increasing the dose and reducing the interval for patients with Crohn's disease. Also, the efficacy of infliximab for both diseases is suggested to be similar. In conclusion, our results show a possible modeling scenario that can accommodate the clinical response to infliximab administered for ulcerative colitis and Crohn's disease. Furthermore, it provides confirmation for the present dosage regimens given for these diseases.

Development of a Screening System for Targeting Carriers Using Peptide-Modified Liposomes and Tissue Sections.

Liposomes have been used as targeting carriers for drug delivery systems (DDSs), and the carriers are able to be modified with targeting ligands, such as antibodies and peptides. To evaluate the targetability of DDS carriers modified with a targeting ligand, culture cells expressing the targeting molecules as well as small animals are used. Furthermore, in vitro and in vivo screening analyses must be repeatedly performed. Therefore, it is important to establish an easy and high-precision screening system for targeting carriers. With this aim, we focused that whether this ex vivo system could easily support assessment of interaction between targeting ligand and its receptor under physiological environment and further screen the DDS carrier-modified with targeting moiety. We examined targeting ability via in vitro, ex vivo, and in vivo analyses using integrin αvß3-targeting C16Y-L. For the in vitro analysis, the cellular uptake of C16Y-L was higher than that of control liposomes in colon26 cells. For the ex vivo analysis, we performed an immunohistochemical analysis using colon26 tumor sections. C16Y-L was specifically attached to the tumor sections, as found in the in vitro analysis. Moreover, to evaluate the ex vivo-in vivo correlation, we examined the intratumoral localization of C16Y-L. This result showed that C16Y-L was accumulated not only in the tumor tissue but also in the tumor vasculature after the intravenous injection of C16Y-L, suggesting that the ex vivo peptide-modified liposomal analysis was correlated with the in vivo analysis. Thus, the ex vivo peptide-modified liposomal analysis may be an easy and rapid screening system with high-precision and for consideration in in vivo conditions.

Biological activity of peptide-conjugated polyion complex matrices consisting of alginate and chitosan.

Peptide-conjugated polysaccharide matrices using bioactive laminin-derived peptides are useful biomaterials for tissue and cell engineering. Here, we demonstrate an easy handling preparation method for peptide-polysaccharide matrices using polyion complex with both alginate and chitosan. First, aldehyde-alginate was synthesized by oxidization of alginate using NaIO4 , and then, reacted with Cys-peptides. Next, the peptide-alginate solution was added to a chitosan-coated plate, and the peptide-polyion complex matrices (peptide-PCMs) were prepared. The peptide-PCMs using an integrin αvß3-binding peptide (A99a: ALRGDN, mouse laminin α1 chain 1145-1150) and an integrin α2ß1-binding peptide (EF1XmR: RLQLQEGRLHFXFD, X = Nle, mouse laminin α1 chain 2751-2763) showed strong cell attachment activity in a dose-dependent manner. When we examined the effect of various spacers on the biological activity of A99a-PCM, hydrophobic and long spacers enhanced the cell attachment activity. Further, the A99a-PCM with the spacers strongly promoted neurite outgrowth. The polyion complex method is an easy way to obtain insolubilized matrix and is widely applicable for various polysaccharides. The peptide-PCM is useful as a biomaterial for cell and tissue engineering.

Identification of laminin α5 short arm peptides active for endothelial cell attachment and tube formation.

Laminin-511, a major component of endothelial basement membrane, consists of α5, ß1, and γ1 chains. The short arm region of the α5 chain is a structural feature of endothelial laminins. In this study, we identified active sequences for human umbilical vein endothelial cells (HUVECs) using recombinant proteins and synthetic peptides. The short arm of the α5 chain contains three globular domains [laminin N-terminal globular domain, laminin 4 domain a, and laminin 4 domain b (LN, L4a, and L4b)] and three rod-like elements [laminin epidermal growth factor-like domain a, b, and c (LEa, LEb, and LEc)]. The cell attachment assay using recombinant proteins showed that RGD-independent cell attachment sites were localized in the α5LN-LEa domain. Further, we synthesized 70 peptides covering the amino acid sequences of the α5LN-LEa domain. Of the 70 peptides, A5-16 (mouse laminin α5 230-243: LENGEIVVSLVNGR) potently exhibited endothelial cell attachment activity. An active sequence analysis using N-terminally and C-terminally truncated A5-16 peptides showed that the nine-amino acid sequence IVVSLVNGR was critical for the endothelial cell attachment activity. Cell adhesion to the peptides was dependent on both cations and heparan sulfate. Further, the A5-16 peptide inhibited the capillary-like tube formation of HUVECs with the cells forming small clumps with short tubes. The eight-amino acid sequence EIVVSLVN in the A5-16 peptide was critical to inhibit HUVEC tube formation. This amino acid sequence could be useful for grafts and thus modulate endothelial cell behavior for vascular surgery. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Down-regulation of cell adhesion via rho-associated protein kinase (ROCK) pathway promotes tumor cell migration on laminin-511.

Epithelial cells, both normal and precancerous, stably anchor to basement membranes, whereas malignant tumors pass through them to achieve metastasis. Of basement membrane components, laminin-511 (α5, ß1, γ1; LM-511) has been found to be a major isoform in many adult basement membranes. Several studies have shown that LM-511 promotes not only cell adhesion but also tumor cell migration. Thus, LM-511 can be viewed like two distinct molecules in normal vs. tumor cells; tumor cells seem to be able to alter their response (adhesive vs. migratory) to LM-511. In this study we examined the effects of biologically active molecules on A549 lung adenocarcinoma cell adhesion to LM-511. Of them, phorbol 12-myristate 13-acetate (PMA) induced transition to a rounded cell shape and significantly promoted cell migration on LM-511. The attachment of PMA-treated A549 cells to LM-511 was weaker than that of control cells. PMA-stimulated signaling pathway reduced the binding of integrin α3ß1 to LM-511. Cell migration assays using inhibitors for signal transduction and cytoskeletal organization showed that suppression of cell adhesion via the rho-associated protein kinase (ROCK) pathway promoted tumor cell migration on LM-511. Our results suggest that the ROCK pathway is involved in the transition from static to migratory cell behaviors on LM-511.

Plasma kisspeptin levels in lactational amenorrhea.

The kisspeptin is a neuropeptide to play physiological roles in regulating gonadotropin-releasing hormone secretion in the hypothalamus. In human plasma, the kisspeptin concentration is measured, but gonadotropin-releasing hormone is not. This study aims to understand the physiological roles of the circulating kisspeptin in lactational amenorrhea in humans because prolactin reduces the kisspeptin expression and luteinizing hormone secretion resulting in anovulations in rodent brains. Plasma kisspeptin levels were measured in 11 subjects in lactational amenorrhea and in four cases with pathological amenorrhea by different etiologies for comparison using the enzyme immunoassay specific for human kisspeptin. The plasma kisspeptin levels in the 11 women with lactational amenorrhea were 15.2 ± 2.5 fmol/mL (mean ± SD) which were not significantly different as compared with 16.5 ± 4.8 fmol/mL (mean ± SD) in four age-matched women with menstrual cycles as we reported previously. In the four cases with pathological amenorrhea, their plasma kisspeptin levels were from 5.8 to 13.7 fmol/mL. This study demonstrated that the plasma kisspeptin levels were not totally reduced in lactational or pathological amenorrhea. These results suggest the physiological roles of the circulating kisspeptin are different from the role in the brain.

Effect of spacer length and type on the biological activity of peptide-polysaccharide matrices.

Peptide-polysaccharide matrices can mimic extracellular matrix structure and function and are useful for tissue and cell engineering. The spacer between the peptide and the polysaccharide is important for both peptide conformation and the interaction between the peptide and receptors. Here, the effect of a spacer on the biological activity of peptide-polysaccharide matrices using various lengths of spacers consisting of glycine, ß-alanine, and ε-aminocaproic acid has been examined. Active laminin-derived peptides, including a syndecan-binding peptide (AG73: RKRLQVQLSIRT), an integrin αvß3-binding peptide (A99a: ALRGDN), and an integrin α6ß1-binding peptide (A2G10: SYWYRIEASRTG), were used as the peptide ligands and chitosan was used as a polysaccharide matrix. The spacers did not influence the biological activity of the AG73-chitosan matrix. In contrast, the integrin-binding peptide-chitosan matrices showed spacer-dependent activity. Hydrophobic spacers enhanced the cell attachment activity of the A99a-chitosan matrix. A four-glycine spacer showed the strongest effect for the biological activity of the A2G10-chitosan matrix. These results suggested that spacer-optimization for each peptide is important for designing effective peptide-polysaccharide matrices. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 512-520, 2016.

Clinical Implications of Plasma N-acetyl-seryl-aspartyl-lysyl-proline Level in Stable Kidney Transplant Recipients.

BACKGROUND: N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. Because AcSDKP is partially eliminated in urine, accumulation of AcSDKP due to chronic renal failure may cause anemia. However, the status of plasma AcSDKP level in stable kidney transplant recipients is unknown although some recipients develop anemia after kidney transplantation. In this study, we investigated the relationship between plasma AcSDKP-like immunoreactive substance (IS) level and clinical characteristics associated with renal anemia in stable kidney transplant recipients. METHODS: Forty Japanese kidney transplant recipients who underwent transplantation more than 90 days prior to the study were included. Morning blood samples were collected and plasma AcSDKP-IS levels were measured using an enzyme immunoassay. RESULTS: A significant correlation was observed between plasma AcSDKP-IS level and creatinine clearance. On the other hand, no significant correlation was observed between plasma AcSDKP-IS level and prolyl oligopeptidase activity, angiotensin II, or erythropoietin level. A significant difference in plasma AcSDKP-IS level was observed between recipients with no renal anemia and those with renal anemia. CONCLUSIONS: These results suggest that plasma AcSDKP level may depend largely on renal function and suggest a possibility that accumulation of AcSDKP may be partially involved in the pathogenesis of renal anemia in stable kidney transplant recipients.

The relationship between circulating kisspeptin and sexual hormones levels in healthy females.

The kisspeptin (metastin) is an endogenous peptide, which regulates human reproduction by modulating gonadotropin-releasing hormone (GnRH) secretion. Kisspeptin was detected in peripheral blood, although GnRH was not. Previously, we measured plasma kisspeptin levels in male healthy subjects and patients with hypogonadism using enzyme immunoassay (EIA) to elucidate a normal range in healthy males and clinical implications of kisspeptin in male hypogonadism. We suggested that the plasma kisspeptin levels were received feedback from testosterone. In this study, we focused female subjects and elucidated the relationship between menstrual cycle and plasma kisspeptin levels to understand kisspeptin-hypothalamic-pituitary-gonadal axis. We measured plasma kisspeptin levels in eight female volunteers. The plasma kisspeptin levels in female are significantly higher than those in male. There are no significant correlation between plasma kisspeptin levels and sexual hormones. We revealed that the kisspeptin might stimulate a start of menstruation as a trigger, and progress menstruation covered for weakened ovarian function. We suggest that kisspeptin may be closely related with menstrual cycle and that the measurement of plasma kisspeptin levels is useful for understanding of reproductive system.

Soluble Lutheran/basal cell adhesion molecule is detectable in plasma of hepatocellular carcinoma patients and modulates cellular interaction with laminin-511 in vitro.

Lutheran (Lu), an immunoglobulin superfamily transmembrane receptor, is also known as basal cell adhesion molecule (B-CAM). Lu/B-CAM is a specific receptor for laminin α5, a subunit of laminin-511 (LM-511) that is a major component of basement membranes in various tissues. Our previous study showed that Lu/B-CAM was cleaved by MT1-MMP and released from cell surfaces. In this study we examined the soluble Lu/B-CAM in culture media and in plasma of mice bearing HuH-7 hepatocellular carcinoma (HCC) cells and patients with HCC. Two HCC cell lines, HepG2 and HuH-7, released Lu/B-CAM into the culture media. Although Lu/B-CAM was cleaved by MT1-MMP in HuH-7 cells, HepG2 cells released Lu/B-CAM in a MMP-independent manner. The concentration of Lu/B-CAM released into mouse plasma correlated with tumor size. Moreover the soluble Lu/B-CAM in plasma of HCC patients was significantly decreased after resection of the tumor. Immunohistochemical studies showed that although the expression of Lu/B-CAM was observed in most HCCs, MT1-MMP was not always expressed in tumor tissues, suggesting that a part of Lu/B-CAM in plasma of HCC patients was also released in a MMP-independent manner. In vitro studies showed that the soluble Lu/B-CAM released from HCC cells bound to LM-511. Moreover the soluble Lu/B-CAM influenced cell migration on LM-511. These results suggest that soluble Lu/B-CAM serves as not only a novel marker for HCC but also a modulator in tumor progression.

Biological activities of the homologous loop regions in the laminin α chain LG modules.

Each laminin α chain (α1-α5 chains) has chain-specific diverse biological functions. The C-terminal globular domain of the α chain consists of five laminin-like globular (LG1-5) modules and plays a critical role in biological activities. The LG modules consist of a 14-stranded ß-sheet (A-N) sandwich structure. Previously, we described the chain-specific biological activities of the loop regions between the E and F strands in the LG4 modules using five homologous peptides (G4EF1-G4EF5). Here, we further analyze the biological activities of the E-F strands loop regions in the rest of LG modules. We designed 20 homologous peptides (approximately 20 amino acid length), and 17 soluble peptides were used for the cell attachment assay. Thirteen peptides promoted cell attachment activity with different cell morphologies. Cell attachment to peptides G1EF1, G1EF2, G2EF1, G3EF4, and G5EF4 was inhibited by heparin, and peptides G1EF1, G1EF2, and G2EF1 specifically bound to syndecan-overexpressing cells. Cell attachment to peptides G2EF3, G3EF1, G3EF3, G5EF1, G5EF3, and G5EF5 was inhibited EDTA. Further, cell attachment to peptides G3EF3, G5EF1, and G5EF5 was inhibited by both anti-integrin α2 and ß1 antibodies, whereas cell attachment to peptide G5EF3 was inhibited by only anti-integrin ß1 antibody. Cell attachment to peptides G1EF4, G3EF4, and G5EF4 was inhibited by both heparin and EDTA and was not inhibited by anti-integrin antibodies. The active peptide sequence alignments suggest that the syndecan-binding peptides contain a "basic amino acid (BAA)-Gly-BAA" motif in the middle of the molecule and that the integrin-binding peptides contain an "acidic amino acid (AAA)"-Gly-BAA motif. Core-switched peptide analyses suggested that the "BAA-Gly-BAA" motif is critical for binding to syndecans and that the "AAA-Gly-BAA" motif has potential to recognize integrins. These findings are useful for understanding chain-specific biological activities of laminins and to evaluate receptor-specific binding mechanisms.

The lutheran/basal cell adhesion molecule promotes tumor cell migration by modulating integrin-mediated cell attachment to laminin-511 protein.

Cell-matrix interactions are critical for tumor cell migration. Lutheran (Lu), also known as basal cell adhesion molecule (B-CAM), competes with integrins for binding to laminin α5, a subunit of LM-511, a major component of basement membranes. Here we show that the preferential binding of Lu/B-CAM to laminin α5 promotes tumor cell migration. The attachment of Lu/B-CAM transfectants to LM-511 was slightly weaker than that of control cells, and this was because Lu/B-CAM disturbed integrin binding to laminin α5. Lu/B-CAM induced a spindle cell shape with pseudopods and promoted cell migration on LM-511. In addition, blocking with an anti-Lu/B-CAM antibody led to a flat cell shape and inhibited migration on LM-511, similar to the effects of an activating integrin ß1 antibody. We conclude that tumor cell migration on LM-511 requires that Lu/B-CAM competitively modulates cell attachment through integrins. We suggest that this competitive interaction is involved in a balance between static and migratory cell behaviors.

Screening of integrin-binding peptides in a laminin peptide library derived from the mouse laminin ß chain short arm regions.

Laminins, major components of basement membrane, consist of three different subunits, α, ß, and γ chains, and so far, five α, three ß, and three γ chains have been identified. We have constructed synthetic peptide libraries derived from the laminin sequences and identified various cell-adhesive peptides. Ten active peptides from the laminin α chain sequences (α1-α5) were found to promote integrin-mediated cell adhesion. Previously, we found fourteen cell-adhesive peptides from the ß1 chain sequence but their receptors have not been analyzed. Here, we expanded the synthetic peptide library to add peptides from the short arm regions of the laminin ß2 and ß3 chains and screened for integrin-binding peptides. Twenty-seven peptides promoted human dermal fibroblast (HDF) attachment in a peptide-coated plate assay. The morphological appearance of HDFs on the peptide-coated plates differed depending on the peptides. B34 (REKYYYAVYDMV, mouse laminin ß1 chain, 255-266), B67 (IPYSMEYEILIRY, mouse laminin ß1 chain, 604-616), B2-105 (APNFWNFTSGRG, mouse laminin ß2 chain, 1081-1092), and B3-19 (GHLTGGKVQLNL, mouse laminin ß3 chain, 182-193) promoted HDF spreading and HDF attachment was inhibited by EDTA, suggesting that the peptides interact with integrins. Immunostaining analyses revealed that B67 induced well-organized actin stress fibers and focal contacts containing vinculin, however, B34, B2-105, and B3-19 did not exhibit stress fiber formation or focal contacts. The inhibition assay using anti-integrin antibodies indicated that B67 interacts with α3, α6, and ß1 integrins, and B34 and B3-19 interact with ß1 integrin. Based on adhesion analysis of peptides modified with an alanine scan and on switching analysis with the homologous inactive sequence B2-64 (LPRAMDYDLLLRW, mouse laminin ß2 chain, 618-630), the Glu(8) residue in the B67 peptide was critical for HDF adhesion. These findings are useful for identifying an integrin binding motif. The B67 peptide has potential for use as a molecular probe for integrins.

Double-stranded DNA stereoselectively promotes aggregation of amyloid-like fibrils and generates peptide/DNA matrices.

An amyloidogenic LAM-L peptide (AASIKVAVSADR, all-L configuration) derived from laminin promoted cell adhesion, neurite outgrowth, and angiogenesis. Here, we prepared novel matrices using double-stranded DNA and the LAM-L peptide. Double-stranded DNA promoted aggregation of amyloid-like fibrils and generated a LAM-L/DNA matrix through electrostatic interactions between the phosphate groups of DNA and the amino groups of LAM-L. This formation of peptide/DNA matrix depends on the Ile-Lys-Val-Ala-Val (IKVAV) sequence in the peptide, since LAM-RM peptide (AASVVIAKSADR), which is scrambled peptide of LAM-L, did not form a matrix with DNA. Further, LAM-D (all-D configuration of LAM-L), which forms amyloid-like fibrils and promotes similar biological activities as LAM-L, did not form amyloid-like fibrils with DNA, suggesting that DNA selectively interacts with the L-configured peptide. Moreover, the LAM-L/DNA matrices showed stronger cell attachment activity compared with LAM-L alone, suggesting the LAM-L/DNA matrices have potential for use as a novel biomaterial in tissue engineering.

Significant decrease in plasma N-acetyl-seryl-aspartyl-lysyl-proline level in patients with end stage renal disease after kidney transplantation.

N-acetyl-seryl-aspartyl-lysyl-proline (AcSDKP) is an endogenous peptide released from its precursor (thymosin-ß4) by prolyl oligopeptidase. AcSDKP is a natural inhibitor of pluripotent hematopoietic stem cell proliferation and is normally found in human plasma. AcSDKP has been shown to be a potent angiogenic factor and to suppress renal fibroblast proliferation. Impairment of renal function has been suggested to have a significant impact on plasma AcSDKP level. The aim of this study was to assess whether improvement of renal function after kidney transplantation has an impact on plasma AcSDKP-like immunoreactive substance (IS) level. Fourteen patients with end stage renal disease (ESRD) who were scheduled to undergo the first kidney allograft transplantation were enrolled. Plasma AcSDKP-IS levels were measured before and 3, 7, 10, 14, 21, 30, 60 and 90 d after kidney transplantation. Plasma AcSDKP-IS level decreased significantly from day 3 after kidney transplantation compared to before kidney transplantation. Creatinine clearance increased significantly from day 7 after kidney transplantation. A significant negative correlation was observed between creatinine clearance and plasma AcSDKP-IS level from before transplantation to 90 d after kidney transplantation. Stepwise multiple regression analysis identified creatinine clearance as the only significant independent factor associated with plasma AcSDKP-IS levels. These results suggest that recovery of kidney function after kidney transplantation may lead to a decrease in plasma AcSDKP level in patients with ESRD, and that plasma AcSDKP level may depend largely on renal function.

Plasma kisspeptin levels in male cases with hypogonadism.

The hypothalamic hormone kisspeptin (metastin) regulates human reproduction by modulating gonadotropin-releasing hormone (GnRH) secretion. Kisspeptin is detected in peripheral blood, although GnRH is not. In this study, we measured plasma kisspeptin levels in four male cases with hypogonadism and seven normal male controls using enzyme immunoassay (EIA) to elucidate the clinical implications of kisspeptin levels in male hypogonadism. The results showed a variety of plasma kisspeptin levels: 6.0 fmol/mL in a male with isolated hypogonadotropic hypogonadism (IHH), 43.2 fmol/mL in a male with Kallmann's syndrome, 40.7 fmol/mL in a male with azoospermia, 323.2 fmol/mL in a male with hypergonadotropic hypogonadism, and 12.3 ± 2.5 fmol/mL (mean ± SD) in seven normal controls. Except for the case with IHH, the plasma kisspetin levels were elevated in the three cases with Kallmann's syndrome, azoospermia, and hypergonadotropic hypogonadism. The reason why the three cases had high values was their lesions were downstream of the kisspeptin neuron in the hypothalamic-pituitary-gonadal axis, suggesting that elevated kisspeptin levels were implicated in hypothalamic kisspeptin secretion under decreased negative feedback of gonadal steroids. The result that the plasma kisspeptin levels were decreased by gonadotropin therapy in the case with Kallmann's syndrome supported this hypothesis. In conclusion, to measure plasma kisspeptin levels could be useful for better understanding of male hypogonadism.

Plasma kisspeptin levels in male cases with hypogonadism.

The hypothalamic hormone kisspeptin (metastin) regulates human reproduction by modulating gonadotropin-releasing hormone (GnRH) secretion. Kisspeptin is detected in peripheral blood, although GnRH is not. In this study, we measured plasma kisspeptin levels in four male cases with hypogonadism and seven normal male controls using enzyme immunoassay (EIA) to elucidate the clinical implications of kisspeptin levels in male hypogonadism. The results showed a variety of plasma kisspeptin levels: 6.0 fmol/ml in a male with isolated hypogonadotropic hypogonadism (IHH), 43.2 fmol/ml in a male with Kallmann's syndrome, 40.7 fmol/ml in a male with azoospermia, 323.2 fmol/ml in a male with hypergonadotropic hypogonadism, and 12.3 ± 2.5 fmol/ml (mean ± SD) in seven normal controls. Except for the case with IHH, the plasma kisspetin levels were elevated in the three cases with Kallmann's syndrome, azoospermia, and hypergonadotropic hypogonadism. The reason why the three cases had high values was their lesions were downstream of the kisspeptin neuron in the hypothalamic-pituitary-gonadal axis, suggesting that elevated kisspeptin levels were implicated in hypothalamic kisspeptin secretion under decreased negative feedback of gonadal steroids. The result that the plasma kisspeptin levels were decreased by gonadotropin therapy in the case with Kallmann's syndrome supported this hypothesis. In conclusion, to measure plasma kisspeptin levels could be useful for better understanding of male hypogonadism.

Identification of cell adhesive sequences in the N-terminal region of the laminin α2 chain.

The laminin α2 chain is specifically expressed in the basement membrane surrounding muscle and nerve. We screened biologically active sequences in the mouse laminin N-terminal region of α2 chain using 216 soluble peptides and three recombinant proteins (rec-a2LN, rec-a2LN+, and rec-a2N) by both the peptide- or protein-coated plate and the peptide-conjugated Sepharose bead assays. Ten peptides showed cell attachment activity in the plate assay, and 8 peptides were active in the bead assay. Seven peptides were active in the both assays. Five peptides promoted neurite outgrowth with PC12 cells. To clarify the cellular receptors, we examined the effects of heparin and EDTA on cell attachment to 11 active peptides. Heparin inhibited cell attachment to 10 peptides, and EDTA significantly affected only A2-8 peptide (YHYVTITLDLQQ, mouse laminin α2 chain, 117-128)-mediated cell attachment. Cell attachment to A2-8 was also specifically inhibited by anti-integrin ß1 and anti-integrin α2ß1 antibodies. These results suggest that A2-8 promotes an integrin α2ß1-mediated cell attachment. The rec-a2LN protein, containing the A2-8 sequence, bound to integrin α2ß1 and cell attachment to rec-a2LN was inhibited by A2-8 peptide. Further, alanine substitution analysis of both the A2-8 peptide and the rec-a2LN+ protein revealed that the amino acids Ile-122, Leu-124, and Asp-125 were involved in integrin α2ß1-mediated cell attachment, suggesting that the A2-8 site plays a functional role as an integrin α2ß1 binding site in the LN module. These active peptides may provide new insights on the molecular mechanism of laminin-receptor interactions.