Induction of differentiation in a human promyelocytic leukemic cell line (HL-60). Production of granule proteins

Serum fibronectin inhibits the adhesion of neutrophil granulocytes (PMNs) to clean glass, HSA-coated glass, and gelatin-coated glass. It does not affect adhesion to collagen-coated glass which itself provides a substratum of low adhesiveness for PMNs. Cell-cell adhesion is not affected. During the acute inflammatory response in vivo, PMNs must migrate through the fibronectin and collagen containing extracellular matrix: reducing cell-substratum adhesion in these circumstances might facilitate locomotion towards inflammatory foci.     

Solution structure of a synthetic peptide corresponding to a receptor binding region of FSH (hFSH-beta 33-53).

The receptor binding surface of human follicle-stimulating hormone (hFSH) is mimicked by synthetic peptides corresponding to the hFSH- chain amino acid sequences 33–53 [Santa-Coloma, T. A., Dattatreyamurty, D., and Reichert, L. E., Jr. (1990),Biochemistry 29, 1194–1200], 81–95 [Santa-Coloma, T. A., and Reichert, L. E., Jr. (1990),J. Biol. Chem. 265, 5037–5042], and the combined sequence (33–53)–(81–95) [Santa-Coloma, T. A., Crabb, J. W., and Reichert, L. E., Jr. (1991),Mol. Cell. Endocrinol. 78, 197–204]. These peptides have been shown to inhibit binding of hFSH to its receptor. Circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy were used to determine the structure of the first peptide in this series, the 21 amino acid peptide hFSH--(33–53), H₂N-YTRDLVYKDPARPKIQKTCTF-COOH. Analysis of CD data indicated the presence of approximately equal amounts of antiparallel -pleated sheet, turns including a -turn, other structures, and a small amount ofa-helix. The major characteristics of the structure were found to be relatively stable at acidicpH and the predominant effect of increased solvent polarity was a small increase ina-helical content. One- and two-dimensional NMR techniques were used to obtain full proton and carbon signal assignments in aqueous solution atpH 3.1. Analysis of NMR results confirmed the presence of the structural features revealed by CD analysis and provided a detailed picture of the secondary structural elements and global folding pattern in hFSH--(33–53). These features included an antiparallel -sheet (residues 38–51 and 46–48), turns within residues 41–46, and 50–52 (a -turn) and a small N-terminal helical region comprised of amino acids 34–36. One of the turns is facilitated by prolines 42 and 45. Proline-45 was constrained to thetrans conformation, whereas proline-42 favored thetrans conformer (70%) over thecis (30%). Two resonances were observed for the single alanine residue (A-43) sequentially proximal to P-42, but the rest of the structure was minimally affected by the isomerization at proline-42. The major population of molecules, containingtrans-42 andtrans-45 prolines, presented 120 NOEs. Distance geometry calculations with 140 distance constraints and energy minimization refinements were used to derive a moderately well-defined model of the peptide's structure. The hFSH--(33–53) structure has a highly polar surface composed of six cationic amino acid (arginie-35, lysine-40, arginine-44, lysine-46, glutamine-48, and lysine-49) and two anionic residues (aspartate-36 and aspartic acid-41). A hydrophobic region in the structure is composed of residues in the antiparallel -sheet and -turn which fold to produce a distorted hairpin. The structure of this domain, together with the protruding and positively charged region in the vicinity of residues 42–45, may mimic the surface of hFSH that binds to the receptor.Abreviations used: hFSH, human follicle-stimulating hormone; PB, 25 mM Na₂KPO₄, 25 mM KH₂PO₄, and 5 mM Mg Cl₂; CD, circular dichroism spectrapolarimetry; NMR, nuclear magnetic resonance spectrometry; COSY, homonuclear correlated spectroscopy; NOESY, 2D nuclear Overhauser effect spectroscopy; HOHAHA, homonuclear Hartman-Han coherence transfer; HMQCHY, reverse-detected heteronuclear multiple shift correlation, one bond; HMBC, reverse-detected heteronuclear multiple bond correlation; S/N, signal to noise ratio; TFE, trifluoroethanol.Dr. Santa-Coloma is on leave of absence from the National Research Council of Argentina (CONICET).     

Anti-apoptotic role of focal adhesion kinase (FAK). Induction of inhibitor-of-apoptosis proteins and apoptosis suppression by the overexpression of FAK in a human leukemic cell line, HL-60

Focal adhesion kinase (FAK) has an anti-apoptotic role in anchorage-dependent cells via an unknown mechanism. To elucidate the role of FAK in anti-apoptosis, we have established several FAK cDNA-transfected HL-60 cell lines and examined whether FAK-transfected cells have resistance to apoptotic stimuli. FAK-transfected HL-60 (HL-60/FAK) cells were highly resistant to apoptosis induced with hydrogen peroxide (1 mm) and etoposide (50 microg/ml) compared with the parental HL-60 cells or the vector-transfected cells, when determined using viability assay, DNA fragmentation, and flow cytometry analysis. Because no proteolytic cleavage of pro-caspase 3 to mature caspase 3 fragment was observed in HL-60/FAK cells, FAK was presumed to inhibit an upstream signal pathway leading to the activation of caspase 3. HL-60/FAK activated the phosphatidylinositide 3'-OH-kinase-Akt survival pathway and exhibited significant activation of NF-kappaB with marked induction of inhibitor-of-apoptosis proteins (IAPs: cIAP-1, cIAP-2, XIAP), regardless of the hydrogen peroxide-treated or untreated conditions, whereas no significant IAPs were detected in the parental or vector-transfected HL-60 cells. Apoptotic agents induced higher NF-kappaB activation in HL-60/FAK cells than in HL-60/Vect cells, and it appeared that sustained NF-kappaB activation is critical to the anti-apoptotic states in HL-60/FAK cells. Mutagenesis of FAK cDNA revealed that Y397 and Y925, which are involved in the tyrosine-phosphorylation sites, were prerequisite for the anti-apoptotic activity as well as induction of IAPs, and that K454, which is involved in the kinase activity, was also required for the full anti-apoptotic activity of FAK. Taken together, we have demonstrated definitively that FAK-transfected HL-60 cells, otherwise sensitive to apoptosis, become resistant to the apoptotic stimuli. We conclude that FAK activates the phosphatidylinositide 3'-OH-kinase-Akt survival pathway with the concomitant activation of NF-kB and induction of IAPs, which ultimately inhibit apoptosis by inhibiting caspase-3 cascade.     

Protein kinase C activity during sphinganine potentiation of retinoic acid-induced differentiation in a human leukemia cell line (HL-60).

The differentiation of HL-60 promyelocytic cells toward mature granulocytic cells induced by retinoic acid (RA) was accompanied by a decrease in protein kinase C (PKC) activity. The enhancement of RA-induced differentiation and the potentiation of the decrease of PKC activity by sphinganine (SP) seemed to correlate with each other. Kinetically, PKC activity during RA-induced differentiation without SP decreased to its lowest (75% of the control) after 48h; about 50% of the reduction was observed at 24h. In the presence of SP, PKC activity decreased more rapidly to its lowest (60% of the control) within 24h of incubation of RA. SP, added 24h before or concomitantly with the addition of RA, could potentiate the RA-induced differentiation and the reduction of PKC activity. Our results indicate that the effect of SP and the role of PKC during RA-induced differentiation may be critical at the early stages of induction of differentiation (within 24h of RA exposure).     

cDNA array analysis of alterations in gene expression in the promyelocytic leukemia cell line,HL-60, after apoptosis induction with etoposide

Alterations in gene expression during apoptosis in HL-60 cells were identified by a cDNA based array analysis. Apoptosis was induced in the human promyelocytic leukemia cell line, HL-60, by incubation with 30 M etoposide for 5 hours. Changes in gene expression occurring during apoptosis in these cells were detected using the ATLAS cDNA Expression Array technique. 40 genes were identified as differentially expressed in the apoptotic cells by at least a factor of two. 30 of these genes were down-regulated during apoptosis. Many of the down-regulated genes reflected decreased proliferative activity in the cells as well as decreased activity of survival pathways. Most of the genes, which were up-regulated during apoptosis, were genes involved in pathways leading to cell death and suppression of proliferation. Based on the up-regulations observed at the mRNA level, it is speculated that etoposide-induced apoptosis in the HL-60 cells proceeds via pathways involving factors such as TNF, IGFBP3, SAPK1, AP-1 and GADD153/CHOP10. Four genes, which showed changes at the mRNA level, were also analyzed by Western blotting in order to confirm the observed differences at the protein level.     

Novel cytotoxic exhibition mode of antimicrobial peptide anoplin in MEL cells,the cell line of murine Friend leukemia virus‐induced leukemic cells

Anoplin is a recently discovered antimicrobial peptide (AMP) isolated from the venom sac of the spider wasp Anoplius samariensis, and it is one of the shortest α‐helical AMP found naturally to date consisting of only ten amino acids. Previous results showed that anoplin exhibits potent antimicrobial activity but little hemolytic activity. In this study, we synthesized anoplin, studied its cytotoxicity in Friend virus‐induced leukemia cells [murine erythroleukemia (MEL) cells], and proposed its possible mechanism. Our results showed that anoplin could inhibit the proliferation of MEL cells in a dose‐dependent and time‐dependent manner via disrupting the integrity of cell membrane, which indicated that anoplin exerts its cytotoxicity efficacy. In addition, the cell cycle distribution of MEL cells was arrested in the G₀/G₁ phase significantly. However, anoplin could not induce obvious apoptosis in MEL cells, as well as anoplin could not induce visible changes on morphology and quantity in the bone marrow cells isolated from normal mice. All of these results indicate that anoplin, as generally believed, is a selective AMP, a value characteristic in the design of safe therapeutic agents. The cytotoxicity of anoplin on MEL cells was mainly attributable to the plasma membrane perturbation and also to the intracellular events such as the arrest of cell cycle. Although this is an initial study that explored the activity of anoplin in vitro rather than in vivo, with the increasing resistance of conventional chemotherapy, there is no doubt that anoplin has desirable feature to be developed as a novel and selective anticancer agent. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Involvement of mitochondria in endoplasmic reticulum stress-induced apoptotic cell death pathway triggered by the prion peptide PrP(106-126)

Prion disorders are progressive neurodegenerative diseases characterized by extensive neuronal loss and by the accumulation of the pathogenic form of prion protein, designated PrP^Sc. Recently, we have shown that PrP_106–126 induces endoplasmic reticulum (ER) stress, leading to mitochondrial cytochrome c release, caspase 3 activation and apoptotic death. In order to further clarify the role of mitochondria in ER stress-mediated apoptotic pathway triggered by the PrP peptide, we investigated the effects of PrP_106–126 on the Ntera2 human teratocarcinoma cell line that had been depleted of their mitochondrial DNA, termed NT2 ρ0 cells, characterized by the absence of functional mitochondria, as well as on the parental NT2 ρ+ cells. In this study, we show that PrP_106–126 induces ER stress in both cell lines, given that ER Ca²⁺ content is low, glucose-regulated protein 78 levels are increased and caspase 4 is activated. Furthermore, in parental NT2 ρ+ cells, PrP_106–126-activated caspase 9 and 3, induced poly (ADP-ribose) polymerase cleavage and increased the number of apoptotic cells. Dantrolene was shown to protect NT2 ρ+ from PrP_106–126-induced cell death, demonstrating the involvement of Ca²⁺ release through ER ryanodine receptors. However, in PrP_106–126-treated NT2 ρ0 cells, apoptosis was not able to proceed. These results demonstrate that functional mitochondria are required for cell death as a result of ER stress triggered by the PrP peptide, and further elucidate the molecular mechanisms involved in the neuronal loss that occurs in prion disorders.     

Molecular characterization of calcitonin gene-related peptide (CGRP) in a rat medullary thyroid carcinoma cell line

The rat medullary thyroid carcinoma cell line, CA-77, is known to express the calcitonin gene and the cell line has been used for characterization of procalcitonin. The present investigations concentrate on a molecular characterization of the calcitonin gene-related peptide (CGRP) expressed by a subclone of this cell line. The investigations demonstrate that this subclone produces significantly more CGRP compared to calcitonin. Gel chromatography of cell extracts demonstrates heterogeneity for both CGRP and calcitonin, but a significant amount of immunoreactivity elutes corresponding to the elution position for synthetic CGRP and calcitonin, respectively. The gel chromatogram for CGRP demonstrates four immunoreactive peaks with K_d of 0.42, 0.53, 0.68, and 0.85. The immunoreactive peak with K_d 0.42 elutes corresponding to synthetic rat CGRP. The four immunoreactive peaks were characterized by high pressure liquid chromatography followed by sequence analysis and mass spectrometry. The immunoreactive peak with K_d 0.42 was identified as rat -CGRP as was the peak with K_d 0.53. The peak with K_d 0.68 was identified as 19–37 rat -CGRP and the peak with K_d 0.85 as 28–37 rat -CGRP. In summary, we find that the CA-77 cell line expresses large quantities of normally processed amidated -CGRP and specific fragments thereof. However, the cell line does not express detectable levels of rat β-CGRP. The findings indicate that the CA-77 cell line can be useful for studies of calcitonin/CGRP gene expression.     

High resolution structure of streptavidin in complex with a novel high affinity peptide tag mimicking the biotin binding motif

A novel peptide was designed which possesses nanomolar affinity of less than 20 nM for streptavidin. Therefore it was termed Nano-tag and has been used as an affinity tag for recombinant proteins. The minimized version of the wild type Nano-tag is a seven-amino acid peptide with the sequence fMDVEAWL. The three-dimensional structure of wild type streptavidin in complex with the minimized Nano-tag was analyzed at atomic resolution of 1.15 A and the details of the binding motif were investigated. The peptide recognizes the same pocket of streptavidin where the natural ligand biotin is bound, but the peptide requires significantly more space than biotin. Therefore the binding loop adopts an "open" conformation in order to release additional space for the peptide. The conformation of the bound Nano-tag corresponds to a 3(10) helix. However, the analysis of the intermolecular interactions of the Nano-tag with residues of the binding pocket of streptavidin reveals astonishing similarities to the biotin binding motif. In principle the three-dimensional conformation of the Nano-tag mimics the binding mode of biotin. Our results explain why the use of the Nano-tag in fusion with recombinant proteins is restricted to their N-terminus and we describe the special significance of the fMet residue for the high affinity binding mode.     

Development of cytochrome b and an active oxidase system in association with maturation of a human promyelocytic (HL-60) cell line

The human HL-60 myeloid leukaemia cell line developed, during maturational changes induced by dimethyl sulphoxide, an enhanced capacity for phorbol myristate acetate- stimulated oxidative activity and acquired a cytochrome b. Titration of the absorbance at 559 nm at potentials of-190 to -370 mV indicated that this cytochrome had a very low potential, differentiating it from mitochondrial and endoplasmic reticulum cytochromes and identifying it as the cytochrome b(-245) that has been recently found in other phagocytic cells. Subcellular fractionation studies of mature HL-60 cells showed that cytochrome b had a dual distribution within the cell. The lighter peak of activity was associated with the plasma membrane markers, adenylate cyclase and receptors for the N- formal-L-methionyl-L-leucyl-L-phenylalanine (f-Met-Leu-Phe) peptide. The denser components localized with the mitochondria but were distinct from mitochondrial cytochromes because whereas the activity of cytochrome c oxidase fell during HL-60 cell maturation, that of this cytochrome b was markedly increased. Concentrations of myeloperoxidase were unrelated to activity of the oxidase system and decreased as the cell matured. The increase in the concentrations of cytochrome b with cellular maturation parallelled the increase in the stimulated nonmitochondrial respiratory activity of these cells. The turnover of the hexose monophosphate shunt of immature cells was increased by the oxidising agents, methylene blue and tert-butylhydroperoxide, indicating that these immature cells have stimulated nonmitochondrial respiratory activity by maturing HL-60 cells is associated with, and is probably dependent upon, the acquisition by these cells of the cytochrome b(-245) oxidase system.     

Decrease in CD93 (C1qRp) expression in a human monocyte-like cell line (U937) treated with various apoptosis-inducing chemical substances

Human CD93, a receptor for complement component 1, subcomponent q phagocytosis (C1qRp), has been shown to be selectively expressed by cells of a myeloid lineage and was originally reported to be involved in the C1q-mediated enhancement of phagocytosis in innate and adaptive immune responses. The modulation of CD93 expression has been investigated in various cells, particularly in granulocytes and monocytes . We previously reported that a protein kinase C activator (PKC), phorbol myristate acetate (PMA), effectively up-regulated CD93 expression on several cultured cell lines and that its regulation was mainly controlled by a PKC delta-isoenzyme. However, the expression pattern of CD93 in myeloid cells with apoptotic properties remains poorly understood. In this study, we examined the modulation of CD93 expression on a human monocyte-like cell line (U937) treated with various apoptosis-inducing chemical substances : an RNA-synthesis inhibitor, actinomycin D (ActD); a DNA topoisomerase I inhibitor, camptothecin (CPT); a protein-synthesis inhibitor, cycloheximide (CHX); a DNA topoisomerase II inhibitor, etoposide (EPS); and a DNA-synthesis inhibitor, mitomycin C (MMC). Apoptosis was monitored using two-color flow cytometry with Annexin V and 7-amino actinomycin D (7AAD). The above-mentioned substances sufficiently induced the early and late stages of apoptosis, identified as Annexin V positive (+)/7AAD negative (-) cells and Annexin V positive (+)/7AAD positive (+) cells, respectively, in U937 cells after 6 hr of treatment. The modulation of CD93 expression on U937 cells during the early stage of apoptosis, gated as Annexin V positive (+)/7AAD negative (-) cells, was then investigated using a CD93 mAb (mNI-11), originally established in our laboratories, and flow cytometry using a fluorescence-activated cell sorter (FACS). The mean fluorescence intensity (MFI) of the cells that stained positive for CD93 mAb (mNI-11) among the treated U937 cells showed a dramatic decrease in expression. In addition, the expressions of HLA-class I (HLA-A, B, C), HLA-class II (HLA-DR), CD18 (lymphocyte function-associated antigen-1 beta; LFA-1beta) and CD54 (intercellular adhesion molecule-1; ICAM-1) were also markedly decreased on the treated U937 cells identified as Annexin V positive (+)/7AAD negative (-) cells (early stage of apoptosis). Interestingly, the expression patterns of CD93 on the U937 cells treated with the above-mentioned chemical substances closely resembled those of HLA-class I (HLA-A, B, C). An immunoblotting analysis showed that the expression of a surface antigen (molecular size, about 97 kDa) targeted by the CD93 mAb (mNI-11) on the U937 cells treated with various apoptosis-inducing chemical substances had clearly decreased. On the other hand, an enzyme-linked immunoassay (EIA) showed that although PMA-treated U937 cells had strongly secreted soluble CD93 (sCD93) into the culture supernatant, the secretion of sCD93 in the culture supernatant of the U937 cells treated with the above-mentioned chemical substances was not enhanced, compared with that of untreated U937 cells. Importantly, however , the U937 cells with apoptotic properties induced by various apoptosis-inducing chemical substances also rapidly (in 30 min) and strongly secreted sCD93 into the culture supernatant in the presence of PMA. Taken together, these findings indicate that the expression of the CD93 molecule identified by CD93 mAb (mNI-11) is dramatically decreased on U937 cells with apoptotic properties, and that the decrease in CD93 expression on U937 cells treated with apoptosis-inducing chemical substances may be a good model for analyzing the regulation of CD93 expression on apoptotic myeloid cells.     

Mechanism of cell death by 5‐aminolevulinic acid‐based photodynamic action and its enhancement by ferrochelatase inhibitors in human histiocytic lymphoma cell line U937

Photodynamic therapy (PDT) for tumors is based on the tumor‐selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), followed by irradiation with visible light. However, the molecular mechanism of cell death caused by PDT has not been fully elucidated. The 5‐aminolevulinic acid (ALA)‐based photodynamic action (PDA) was dependent on the accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells. PDA induced apoptosis characterized by lipid peroxidation, increase in Bak and Bax/Bcl‐xL, decrease in Bid, membrane depolarization, cytochrome c release, caspase‐3 activation, phosphatidylserine (PS) externalization. PDT‐induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria. These cell death events were enhanced by ferrochelatase inhibitors. These results indicated that ALA‐based‐PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA. Copyright © 2009 John Wiley & Sons, Ltd.     

A peptide derived from the nonreceptor binding region of urokinase plasminogen activator (uPA) inhibits tumor progression and angiogenesis and induces tumor cell death in vivo.

Urokinase plasminogen activator (uPA) plays an important role in the progression of several malignancies including breast cancer. We have identified a noncompetitive antagonist of the uPA-uPAR interaction derived from a nonreceptor binding region of uPA (amino acids 136-143). This 8-mer capped peptide (A6) inhibited breast cancer cell invasion and endothelial cell migration in a dose-dependent manner in vitro without altering cell doubling time. Intraperitoneal administration of A6 resulted in a significant inhibition of tumor growth and suppressed the development of lymph node metastases in several models of breast cancer cell growth and metastasis. Large areas of tumor necrosis and extensive positive staining by TUNEL were observed on histological and immunohistochemical analysis of experimental tumor sections from A6-treated animals. A6 treatment also resulted in a decrease in factor VIII-positive tumor microvessel hot-spots. These results identify a new epitope in uPA that is involved in the uPA-uPAR interaction and indicate that an antagonist based on this epitope is able to inhibit tumor progression by modulating the tumor microenvironment in the absence of direct cytotoxic effects in vivo.     

Mutations on N-terminal region of Taiwan cobra phospholipase A(2) result in structurally distorted effects.

In the present study, three Taiwan cobra PLA(2) variants were prepared by adding an extra N-terminal Met, substituting Asn-1 by Met or deleting the N-terminal heptapeptide. Recombinant PLA(2) mutants were expressed in Escherichia coli (E. coli), and purified to homogeneity by reverse phase HPLC. Fluorescence measurement showed that the hydrophobic character of the catalytic site, the microenvironment of Trp residues and energy transfer from excited Trp to 8-anilinonaphthalene sulfonate (ANS) were affected by N-terminal mutations. An alteration in the structural flexibility of the active site was noted with the mutants lacking the N-terminal heptapeptide or with an extra N-terminal Met added as evidenced by the inability of the two variants to bind with Ba(2+). Moreover, modification of Lys residues and energy transfer within the protein-ANS complex revealed that the Ca(2+)-induced change in the global structure of PLA(2) was different from that in N-terminal variants. Together with the fact that an 'activation network' connects the N-terminus with the active site, our data suggest that mutagenesis on the N-terminal region affects directly the fine structure of the catalytic site, which subsequently transmits its influence in altering the structure outside the active site of PLA(2). Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Influence of Wolbachia infection on antimicrobial peptide gene expressions in a cell line of the silkworm,Bombyx mori (Lepidoptera: Bombycidae)

Wolbachia, a symbiotic bacterium found in a broad range of insects, manipulates host reproduction. In addition to reproductive alterations, Wolbachia may also modify the immune system of host insects to protect them from additional pathogenic infection. We hypothesized that Wolbachia exerts protective effects by activating antimicrobial peptide (AMP) genes. To test this hypothesis, we established immunocompetent cell lines derived from the silkworm, Bombyx mori, which were transinfected with two Wolbachia strains, wKue and wCauB, originating from lepidopteran insects and quantified the expression of four AMP genes, cecropin B, defensin B, attacin, and lebocin 3. The expression was measured in wKue-infected, wCauB-infected, and uninfected cells, before and after treatment with peptidoglycan (PGN) that mimicked a bacterial infection. A two-way ANOVA for each gene showed that both Wolbachia infection and PGN treatment significantly increased the gene expression and their interaction. When treated with PGN, wKue- and wCauB-infected cells showed higher expression of the four AMP genes than those in uninfected cells, suggesting that Wolbachia infection increased the ability of host cells to produce AMPs in response to immune stimulation with PGN. These observations suggest that the two Wolbachia strains have immune priming effects and may protect the host insects from a secondary infection.     

Induction of apoptosis in human leukemia (HL-60) cells by skimmed milk digested with a proteolytic enzyme purified from Saccharomyces cerevisiae

Apoptosis-inducing materials were produced by digesting bovine skimmed milk with cell-free extract of Saccharomyces cereviiae at pH 4.8. An enzyme involved in production of the materials was purified from the cell-free extract by successive column chromatography. The purified enzyme was homogeneous and identified as protease B by analyzing N-terminal amino acid sequence. Characteristics features of apoptosis were observed within 5 h of digested skimmed milk treatment as documented by DNA fragmentation, expression of phosphatidylserine. The inducing factors were recovered in the soluble fraction of 92% ethanol, suggesting that the factors were hydrophilic low molecular weight substances.     

Addition of a peptide fragment on an alpha-helical depsipeptide induces alpha/3(10)-conjugated helix: synthesis, crystal structure, and CD spectra of Boc-Leu-Leu-Ala-(Leu-Leu-Lac)3-Leu-Leu-OEt

The depsipeptide Boc(1)-Leu(2)-Leu(3)-Ala(4)-Leu(5)-Leu(6)-Lac(7)-Leu(8)-Leu(9)-Lac(10)-Leu(11)-Leu(12)-Lac(13)-Leu(14)-Leu(15)-OEt(16) (1) (Boc = tert-butyloxycarbonyl, Lac = L-lactic acid residue) has been synthesized from the peptide Boc-Leu-Leu-Ala-OEt (2) and a depsipeptide, Boc-(Leu-Leu-Lac)(3)-Leu-Leu-OEt (3). Single crystals of 1 were successfully obtained and the structure has been solved by direct methods (such as Sir2002 and Shake-and-Bake). Interestingly, 1 adopts an alpha/3(10)-conjugated helix containing a kink at the junction of peptide and depsipeptide segments, Leu3-Lac7. This is significantly different from the conformation of 3, which has a straight alpha-helical structure with standard phi and psi angles. Microcrystalline CD spectra were also studied to compare structural properties of 1 and 3. The differences between alpha/3(10)- and alpha-helices appear in these CD spectra.     

A sea urchin egg jelly peptide induces a cGMP-mediated decrease in sperm intracellular Ca(2+) before its increase

Speract, a sperm-activating peptide (SAP) from sea urchin eggs, increases the intracellular concentration of Ca²⁺ ([Ca²⁺]i) and modulates sperm motility. We measured the initial sperm response to speract using its caged analog and observed, for the first time, a small but significant decrease in sperm [Ca²⁺]i before the increase. Both directions of the [Ca²⁺]i change were completely blocked in high K⁺ seawater. Using membrane-permeant caged cyclic nucleotides (cNMP), only cGMP induced the decrease in [Ca²⁺]i although both cGMP and cAMP increased the [Ca²⁺]i. The decrease in the [Ca²⁺]i induced by cGMP was more notable following a second photolytic event, once [Ca²⁺]i had been elevated by an initial flash. This pattern of [Ca²⁺]i change was confirmed in individual sperm. These results together with pharmacological evidence suggest that the initial [Ca²⁺]i decrease is due to a Na⁺/Ca²⁺ exchanger activity, stimulated by hyperpolarization mediated by K⁺ efflux through cGMP-regulated K⁺ channels.