Synthesis of potent CXCR4 inhibitors possessing low cytotoxicity and improved biostability based on T140 derivatives.

A peptidic CXCR4 antagonist T140 efficiently blocks the entry of T cell line-tropic strains of HIV-1 (X4-HIV-1) into target cells. In this study, a series of T140 derivatives, replacing the basic amino acid residues with Glu (D-Glu) and/or L-citrulline (Cit), were synthesized in order to reduce non-specific binding and cytotoxicity. Among them, TE14011 ([Cit6, D-Glu8]-T140 with the C-terminal amide) exhibited strong anti-HIV activity and low cytotoxicity. TE14011 was found to be stable in mouse serum, but unstable in rat liver homogenate due to the deletion of the N-terminal Arg1-Arg2-L-3-(2-naphthyl)alanine (Nal)3 residues from the parent peptide. N-Terminal acetylation of TE14011 led to the development of a novel lead compound, Ac-TE 14011, which possesses a high selectivity index as well as increased stability in serum and liver homogenate.

Nuclear mislocalization of enzymatically active RanGAP causes segregation distortion in Drosophila.

Segregation Distorter (SD) is a meiotic drive system in Drosophila that causes preferential transmission of the SD chromosome from SD/SD+ males owing to dysfunction of SD+ spermatids. The Sd locus, which is essential for distortion, encodes a truncated RanGAP (Ran GTPase activating protein), a key nuclear transport factor. Here, we show that Sd-RanGAP retains normal enzyme activity but is mislocalized to nuclei. Distortion is abolished when enzymatic activity or nuclear localization of Sd-RanGAP is perturbed. Overexpression of Ran or RanGEF (Ran GTPase exchange factor) in the male germline fully suppresses distortion. We conclude that mislocalization of Sd-RanGAP causes distortion by reducing nuclear RanGTP, thereby disrupting the Ran signaling pathway. Nuclear transport of a GFP reporter in salivary glands is impaired by SD, suggesting that a defect in nuclear transport may underlie sperm dysfunction.

Effects of fukinolic acid and cimicifugic acids from Cimicifuga species on collagenolytic activity.

The inhibitory collagenolytic activity (47-64% inhibition in 0.22-0.24 microM) of fukinolic acid and cimicifugic acids A, B, and C, which are esters of fukiic acid (3',4'-dihydroxybenzyl tartaric acid) was more potent than that (20-37% inhibition in 0.23-0.24 microM) of cimicifugic acids D, E, F, which are esters of pscidic acid (4'-hydroxybenzyl tartaric acid). Since fukiic acid showed weaker inhibition, and caffeic acid, ferulic acid, isoferulic acid, and p-coumaric acid showed far weaker activities, the entire structures of fukinolic acid and cimicifugic acids A, B, and C proved to be responsible for the inhibitory activities. Trypsin and pronase E hydrolyzed collagen nonselectively alone or in addition to collagenase. These collagenolytic activities were also inhibited by fukinolic acid. These results show that fukinolic acid may inhibit either the collagenolytic activities specific to collagenase or nonspecific to other emzymes. The present studies suggest the potential effect of fukinolic acid and cimicifugic acids of Cimicifuga rhizomes in preventing collagen degradation by collagenases or collagenolytic enzymes under pathological conditions, wound healing, or inflammation.

Many genomic regions are required for normal embryonic programmed cell death in Caenorhabditis elegans.

To identify genes involved in programmed cell death (PCD) in Caenorhabditis elegans, we screened a comprehensive set of chromosomal deficiencies for alterations in the pattern of PCD throughout embryonic development. From a set of 58 deficiencies, which collectively remove approximately 74% of the genome, four distinct classes were identified. In class I (20 deficiencies), no significant deviation from wild type in the temporal pattern of cell corpses was observed, indicating that much of the genome does not contain zygotic genes that perform conspicuous roles in embryonic PCD. The class II deficiencies (16 deficiencies defining at least 11 distinct genomic regions) led to no or fewer-than-normal cell corpses. Some of these cause premature cell division arrest, probably explaining the diminution in cell corpse number; however, others have little effect on cell proliferation, indicating that the reduced cell corpse number is not a direct result of premature embryonic arrest. In class III (18 deficiencies defining at least 16 unique regions), an excess of cell corpses was observed. The developmental stage at which the extra corpses were observed varied among the class III deficiencies, suggesting the existence of genes that perform temporal-specific functions in PCD. The four deficiencies in class IV (defining at least three unique regions), showed unusually large corpses that were, in some cases, attributable to extremely premature arrest in cell division without a concomitant block in PCD. Deficiencies in this last class suggest that the cell death program does not require normal embryonic cell proliferation to be activated and suggest that while some genes required for cell division might also be required for cell death, others are not. Most of the regions identified by these deficiencies do not contain previously identified zygotic cell death genes. There are, therefore, a substantial number of as yet unidentified genes required for normal PCD in C. elegans.

Studies on the constituents of Cimicifuga species. XXVIII. Cycloart-7-enol glycosides from the underground parts of Cimicifuga simplex Wormsk.

Four new cycloart-7-ene triterpenol arabinosides, bugbanosides C-F, were isolated from the underground parts of Cimicifuga simplex Wormsk. (Ranunculaceae). The structures were elucidated as 12beta-acetoxy-3beta,15alpha,-24R,25-tetrahydroxy-16,23-dione-cycloart-7-ene 3-O-alpha-arabinopyranoside, 12beta-acetoxy-24R,25-epoxy-3beta,15alpha-dihydroxy-16,23-dione-cycloart-7-ene 3-O-alpha-L-arabinopyranoside, 12beta-acetoxy-24R,25-epoxy-3beta-hydroxy-16,23-dione-cycloart-7-ene 3-O-alpha-L-arabinopyranoside, and 16,23R:16,24S-diepoxy-3beta,12beta,15alpha,25-tetrahydroxy-cycloart-7-ene 3-O-alpha-L-arabinopyranoside on the basis of spectral and chemical evidence. The circular dichroism (CD) of bugbanosides C-F showed strong negative maxima at 214-217 nm due to a cycloart-7-ene system, as well as other cycloart-7-ene triterpenes. The CD data showed to be useful in determining basic skeletons, including absolute stereostructures of cycloart-7-ene triterpenes.

Truncated RanGAP encoded by the Segregation Distorter locus of Drosophila.

Segregation Distorter (SD) in Drosophila melanogaster is a naturally occurring meiotic drive system in which the SD chromosome is transmitted from SD/SD+ males in vast excess over its homolog owing to the induced dysfunction of SD+-bearing spermatids. The Sd locus is the key distorting gene responsible for this phenotype. A genomic fragment from the Sd region conferred full distorting activity when introduced into the appropriate genetic background by germline transformation. The only functional product encoded by this fragment is a truncated version of the RanGAP nuclear transport protein. These results demonstrate that this mutant RanGAP is the functional Sd product.

Studies on inhibitory activities of fukiic acid esters on germination, alpha-amylase and carboxypeptidase A.

Fukinolic acid (1) and cimicifugic acid A (2), caffeic acid analogs, as well as rosmarinic acid (3) and caffeic acid (4) showed inhibition on seed germination and seedling growth. The potency of 1 and 2 was comparable with that of 3. Compounds 1 and 2 also showed strong inhibitory activities as well as 3 and 4 on alpha-amylase. The activity of 1 was higher than that of acarbose used as a positive control, and its 50% inhibitory concentration (IC50) was 2.41 x 10(-5) M. Compounds 1 and 2 also showed inhibitory activities strong as 3 and stronger than 4 on carboxypeptidase A. The activities of 1 and 2 were higher than that of 1, 10-phenanthroline used as a positive control.

Antimalarial activity and nucleoside transport inhibitory activity of the triterpenic constituents of Cimicifuga spp.

The in vitro antimalarial activity against human malaria parasite (Plasmodium falciparum, FCR-3 strain) was examined using 59 triterpenoids obtained during studies on the triterpenic constituents of Cimicifuga spp. The 50% effective concentration values (EC50) of 25 active triterpenoids were 1.0-3.0 microM, and 19 of the compounds had a common 16, 23:23, 26:24, 25-triepoxy group in the side-chain moieties. Among the active triterpenoids, 9 also showed significant inhibition of nucleoside transport in mouse splenocytes. A relationship between the antimalarial activity and the inhibition of nucleoside transport involving these triterpenoids is discussed.

A protease sensitive region of plant and animal ribonucleases belonging to the RNase T2 family.

Recombinant RNase LE from tomato and squid liver RNase Tp, typical plant/animal type RNases belonging to the RNase T2 family, were subjected to limited digestion with several proteases, and the cleavage sites were analyzed by Edman degradation. Recombinant RNase LE was cleaved specifically at the 24th Lys by lysylendopeptidase and trypsin, and RNase Tp was cleaved at the 21st Glu by V8 protease. These cleavage sites are located very close to those where the cleavage during preparation of several animal RNase T2 family enzymes was observed. From this finding, it was concluded that the short segment around the 20th amino acid residue in plant/animal RNases is located on the surface of the molecules and forms loops, and is thus very sensitive to proteases.

Cellular localization and expression of template-activating factor I in different cell types.

Template-activating factors I (TAF-I) alpha and beta have been identified as chromatin remodeling factors from human HeLa cells. TAF-I beta corresponds to the protein encoded by the set gene, which was found in an acute undifferentiated leukemia as a fusion version with the can gene via chromosomal translocation. To determine the localization of TAF-I, we raised both polyclonal and monoclonal antibodies against TAF-I. The proteins that react to the antibodies are present not only in human cells but also in mouse, frog, insect, and yeast cells. The mouse TAF-I homologue is ubiquitous in a variety of tissue cells, including liver, kidney, spleen, lung, heart, and brain. It is of interest that the amounts of TAF-I alpha and beta vary among hemopoietic cells and some specific cell types do not contain TAF-I alpha. The level of the TAF-I proteins does not change significantly during the cell cycle progression in either HeLa cells synchronized with an excess concentration of thymidine or NIH 3T3 cells released from the serum-depleted state. TAF-I is predominantly located in nuclei, while TAF-I that is devoid of its acidic region, the region which is essential for the TAF-I activity, shows both nuclear and cytoplasmic localization. The localization of TAF-I in conjunction with the regulation of its activity is discussed.

Primary structure of a squid acid and base non-specific ribonuclease.

Squid (Todarodes pacificus) liver RNase (RNase Tp) was purified. RNase Tp was a base non-specific and acid RNase. Upon hydrolysis of RNA, RNase Tp released four mononucleotides in the order of G > A > U > C. RNase Tp consisted of two peptides with 198 and 23 amino acid residues. The amino acid sequences of these peptides were analyzed. The large peptide had two unique segments containing most of the active site amino acid residues of RNase T2 family enzymes. From the comparison of the sequence of short peptide with the sequences of the other RNase belonging to RNase T2 family RNases, it was found that the amino acid sequence of the short peptide was very similar to that of the C-terminal portion of RNases of the RNase T2 family. Thus, we concluded that the short peptide was a C-terminal part of RNase Tp. The molecular mass of the protein moiety of RNase Tp was 25,582 daltons. The amino acid sequence of RNase Tp most resembles that of oyster RNase (91 amino acid residues identical) in the RNase T2 family RNases. However, the N-terminal portion of RNase Tp was unusually similar to those of plant RNases, rather than the other animal RNases.

Panosialins, inhibitors of an alpha1,3-fucosyltransferase Fuc-TVII, suppress the expression of selectin ligands on U937 cells.

Panosialins A and B were isolated as inhibitors of an alpha1,3-fucosyltransferase, Fuc-TVII, which is a key enzyme in the biosynthesis of selectin ligands, from culture broth of Streptomyces sp. Panosialins A and B inhibited the Fuc-TVII activity with IC50 values of 4.8 and 5.3 microg/ml, respectively. Panosialin A suppressed expression of selectin ligands on U937 cells, and inhibited the cell adhesion to immobilized E-selectin-immunoglobulin. Panosialins are the first reported Fuc-TVII inhibitors which can suppress the biosynthesis of selectin ligands and then inhibit selectin-mediated cell adhesion.

Primary structure of porcine spleen ribonuclease: sequence homology.

The primary structure of porcine spleen RNase (RNase Psp1) was investigated as a mean of assessing the structure-function relationship of base non-specific ribonucleases of animal origin. N-terminal analysis of RNase Psp1 yielded three N-terminal sequences. These peptides were separated by gel-filtration on Superdex 75HR, after reduction and S-carboxymethylation of RNase Psp1. Determination of the amino-acid sequence of these peptides indicated that the RNase Psp1 preparation consisted of three peptides having 20 (RCM RNase Psp1 pep1), 15 (RCM RNase Psp1 pep2), and 164 (RCM RNase Psp1 pro) amino-acid residues, respectively. It possessed two unique segments containing most of the active site amino-acid residues of the RNases of the RNase T2 family. The alignment of these three peptides in RNase Psp1 was determined by comparison with the other enzymes in the RNase T2 family. The overall results showed that RCM RNase Psp1 pep1 and RCM RNase Psp1 pep2 are derived from the N-terminal and C-terminal regions of RNase Psp1, respectively, probably by processing by some protease. The molecular mass of the protein moiety of RNase Psp1 was 23235 Da.

Studies on the constituents of Cimicifuga species. XIX. Eight new glycosides from Cimicifuga simplex Wormsk.

Eight new glycosides were isolated from Cimicifuga simplex (Ranunculaceae), and their structures were determined to be 23-O-acetyl-7-8-didehydroshengmanol-3-O-alpha-L-arabinopyranosi de (1), 24-epi-24-O-acetyl-7,-8- didehydrohydroshengmanol-3-O-beta-D-galactopyranoside (2), 7,8-didehydrocimigenol-3-O-beta-D-galacytopyranoside (3), 24-epi-24-O-acetylhydroshengmanol-3-O-beta-D-galactopyranoside (4), cimigenol-3-O-beta-D-galactopyranoside (5), 25-O-methylcimigenol-3-O-beta-D-galactopyranoside (6), 25-O-acetylcimigenol-3-O-beta-D-galactopyranoside (7) and 25-O-acetylcimigenol-3-O-beta-D-glucopyranoside (8). Genuine aglycones were obtained by the hydrolysis of 1--7 with lactase F[Amano] and of 8 with cellulase T[amano]4. Acerinol was prepared from 7,8-didehydrocimigenol and showed antilipemic effects.

Histamine enhanced the TNF-alpha-induced expression of E-selectin and ICAM-1 on vascular endothelial cells.

Cell adhesion molecules are expressed on endothelial cells by various proinflammatory cytokines. Tumor necrosis factor-alpha (TNF-alpha) induces the expression of E-selectin and intercellular adhesion molecule-1 (ICAM-1) on human umbilical vein endothelial cells (HUVEC). Although histamine is a potent vasoactive mediator, it does not induce the expression of E-selectin and ICAM-1. In this report, we show that histamine concentration-dependently enhances the TNF-alpha-induced expression of E-selectin and ICAM-1 on HUVEC. The histamine-enhanced expression of E-selectin and ICAM-1 was inhibited by the histamine H1 receptor antagonists, mepyramine and diphenhydramine. KW-4679 and ketotifen, antiallergic drugs with histamine H1 receptor antagonistic activity, potently inhibit the expression of E-selectin and ICAM-1. A histamine H2 receptor antagonist, ranitidine, did not affect the histamine-induced expression of cell adhesion molecules. These data indicate that histamine induces the expression of E-selectin and ICAM-1 synergistically with TNF-alpha through histamine H1 receptors.

Immunocytochemical study on internalization of anti-carbohydrate monoclonal antibodies.

The fate of anti-tumor monoclonal antibodies (mAbs) after binding to the cell surface is one of the important factors for application of mAbs in therapy. For mAbs to remain on the cell surface for a long time is preferable character for complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC). On the other hand, quick internalization of mAbs into cytoplasm is advantageous for cytotoxic effects of toxins or cytotoxic drugs conjugated with mAbs. In this study we investigated the localization of anti-carbohydrate mAbs in vitro by immunocytochemical methods at the electron microscopic level. Anti-ganglioside GM3 mAb (mouse IgM) was localized both in cytoplasmic vesicles and on the cell surface membrane of mouse melanoma cells after incubation for 30 min at 37 degrees C. Anti-ganglioside GM2 mAb (mouse IgM), anti-ganglioside GD2 mAb (mouse IgG3) anti-ganglioside GD3 mAb (mouse IgG3) mainly existed on the cell surface of human small cell lung carcinoma cells, human neuroblastoma cells and human melanoma cells, respectively, after incubation for 30 min at 37 degrees C. On the other hand, anti-sialyl Lea mAb (mouse IgG1) was intensively incorporated into the cytoplasm of human colon tumor cells under the same conditions. Anti-ganglioside GD3 mAb and anti-sialyl Lea mAb were radiolabelled with 125Iodine and traced in in vitro culture. When the cells were incubated in the presence of the 125I-mAb for 90 min on ice, the ratio of intracellular counts to surface counts was 8/92 for anti-ganglioside GD3 mAb and 31/69 for anti-sialyl Lea mAb respectively. After the subsequent incubation for 60 min at 37 degrees C, the ratio altered to 13/87 for anti-ganglioside GD3 mAb and 54/46 for anti-sialyl Lea antigen mAb. This study suggested that internalization of anti-carbohydrate mAbs after the binding to the cell surface was different among the mAbs depending on the character of antigens. In conclusion, anti-ganglioside mAbs have beneficial characters for CDC or ADCC while anti-sialyl Lea mAb is suitable for immunoconjugates.

Structure of mouse Mx1 protein. Molecular assembly and GTP-dependent conformational change.

Mouse Mx1 protein is an interferon-inducible nuclear protein and confers resistance to influenza virus infection. The Mx1 protein purified from interferon-induced A2G mouse liver exhibited GTPase activity as did the Mx1 protein purified from the Mx1 cDNA-expressing Escherichia coli (Nakayama, M., Nagata, K., Kato, A., and Ishihama, A. (1991) J. Biol. Chem. 266, 21404-21408; Nakayama, M., Nagata, K., and Ishihama, A. (1992) Virus Res. 22, 227-234). The Mx1 protein purified from both mouse liver and Mx1-cDNA expressing E. coli was found to exist as assembled polymeric states judged from gel filtration pattern. By making a set of deletion derivatives of the Mx1 cDNA, the main motif for self-assembly of the Mx1 protein was mapped between amino acid residues 51-99. This motif is highly conserved not only in the Mx family of proteins but also in Mx-related proteins. The polymeric form of Mx1 from E. coli was observed as "horseshoe"-like structure by negative staining microscopy. When the Mx1 protein was incubated with GTP, this horseshoe structure was transformed to larger and tightly stacked helical forms. Electron microscopic analysis of immunostained liver of the interferon-induced mice indicated that the Mx1 protein exists in nuclei, forming giant complexes of about half the size of nucleoli.

Application of anti lung adenocarcinoma monoclonal antibody recognizing cytokeratin-like cytoplasmic antigen for tumor diagnosis.

An anti lung adenocarcinoma murine monoclonal antibody (MoAb), KM195 (IgG1), was generated using mice which underwent tolerance treatment to normal lung tissues. KM195 was selected from among a number of hybridoma clones because of its advantageous reactivity such as high binding to cell membranes of lung adenocarcinoma tissues and low binding to cell membranes of major normal tissues. In a binding assay using cultured cell lines KM195 was found to bind cytoplasmic antigen in many adenocarcinoma cells. Detailed immunohistochemical analysis using paraffin-fixed tissue sections showed that many adenocarcinoma cells such as gastric cancer, colorectal cancer, pancreatic cancer, mammary cancer, ovary cancer and cervical cancer reacted positively with KM195, as well as lung adenocarcinoma cells. KM195 also positively stained a small number of normal cells found in adult and fetal tissues like lung, intestine, pancreas, liver and kidney. Western blot analysis using membrane fraction of lung adenocarcinoma tissues revealed two major KM195-positive bands which were electrophoresed nearby at molecular weights (M.W.) of 40 Kd. The protein corresponding to the two major bands was purified by immuno-affinity chromatography and sequenced. The amino-terminal 19 residues of the lower band was identified as VLEVDPNIQAVXTQEXEQI, which is identical to that of the human cytokeratin 8 (residues 77 to 95), M.W. 52Kd. The amino-terminal sequence of the upper band was blocked and not determined. To examine the ability of KM195 for tumor imaging, 125I-labeled KM195 was injected i.v. into nude mice bearing SW1116 xenografts. Significantly higher radioactivity was observed in the tumor compared with major organs at days 3 and 5. These data indicate that KM195, which recognizes cytokeratin 8-like cytoplasmic antigen, could be a potential MoAb for use in the immunohistochemical diagnosis and radioimmunodetection of adenocarcinoma.