The antisense homology box: a new motif within proteins that encodes biologically active peptides.

Amphiphilic peptides approximately fifteen amino acids in length and their corresponding antisense peptides exist within protein molecules. These regions (termed antisense homology boxes) are separated by approximately fifty amino acids. Because many sense-antisense peptide pairs have been reported to recognize and bind to each other, antisense homology boxes may be involved in folding, chaperoning and oligomer formation of proteins. The antisense homology box-derived peptide CALSVDRYRAVASW, a fragment of human endothelin A receptor, proved to be a specific inhibitor of endothelin peptide (ET-1) in a smooth muscle relaxation assay. The peptide was able to block endotoxin-induced shock in rats as well. Our finding of endothelin receptor inhibitor among antisense homology box-derived peptides indicates that searching proteins for this new motif may be useful in finding biologically active peptides.

In vivo effects of monoclonal antibodies that functionally inhibit complement regulatory proteins in rats.

The present work was designed to evaluate the effects of functional suppression of complement regulatory proteins in vivo. Male Wistar rats were anesthetized with Nembutal and were intravenously injected with 1 mg/kg of F(ab')2 or Fab fraction of either monoclonal antibody 5I2, which inhibits the function of rat counterpart of mouse Crry/p65, or monoclonal antibody 6D1, which inhibits the rat counterpart of CD59. Mean arterial pressure was continuously measured for 30 min. When 5I2 was injected, there was a biphasic change of mean arterial pressure, namely, the rapid increase immediately after the injection (approximately 2 min, phase 1) and the subsequent fall and slow recovery (approximately 4-30 min, phase 2). These effects were completely abrogated by pretreatment of rats with cobra venom factor. Pretreatment with carboxypeptidase inhibitor, which inhibits inactivation of anaphylatoxins C3a and C5a, induced enhanced reduction of blood pressure. Circulating leukocytes and platelets were rapidly decreased 5 min after antibody injection and became normal by 2 h. Hematocrit and erythrocyte count were continuously increased up to 2 h after injection, suggesting that there was hemoconcentration due to increased vascular permeability. Immunofluorescence study revealed binding of antibody fragments and rat C3 along the capillaries of lung, heart, and liver 5 min after injection. In contrast to 5I2, F(ab')2 fraction of 6D1, though localized to the same areas and in similar amounts, had no significant effect on the parameters measured. These data suggest that the rat counterpart of mouse Crry/p65 plays a vital role in vivo by preventing the activation of autologous complement on vascular endothelium.

Changes in the intracranial rheoencephalogram at lower limit of cerebral blood flow autoregulation.

Cerebral blood flow (CBF) reactivity monitoring is an appropriate primary parameter to evaluate cerebral resuscitation due to a systemic or regional cerebral injury leading to possible irreversible brain injury. Use of the electrical impedance method to estimate CBF is rare, as the method's anatomical background is not well understood. Use of intracranial rheoencephalography (iREG) during hemorrhage and comparison of iREG to other CBF measurements have not been previously reported. Our hypothesis was that iREG would reflect early cerebrovascular alteration (CBF autoregulation). Studies comparing iREG, laser Doppler flowmetry and ultrasound were undertaken on anesthetized rats to define CBF changes during hemorrhage. Blood was removed at a rate required to achieve a mean arterial blood pressure (MABP) of 40 mm Hg over 15 min. Estimation of CBF was taken with intracranial, bipolar REG (REG I; n=14), laser Doppler flowmetry (LDF; n=3) and carotid flow by ultrasound (n=11). Data were processed off-line. During the initial phase of hemorrhage, when MABP was close to 40 mm Hg, intracranial REG amplitude transiently increased (80.94%); LDF (77.92%) and carotid flow (52.04%) decreased and changed with systemic arterial pressure. Intracranial REG amplitude change suggests classical CBF autoregulation, demonstrating its close relationship to arteriolar changes. The studies indicate that iREG might reflect cerebrovascular responses more accurately than changes in local CBF measured by LDF and carotid flow. REG may indicate promise as a continuous, non-invasive life-sign monitoring tool with potential advantages over ultrasound, the CBF measurement technique normally applied in clinical practice. REG has particular advantages in non-hospital settings such as military and emergency medicine.

Estrogen receptor immunoreactivity is present in the majority of central histaminergic neurons: evidence for a new neuroendocrine pathway associated with luteinizing hormone-releasing hormone-synthesizing neurons in rats and humans.

The central regulation of the preovulatory LH surge requires a complex sequence of interactions between neuronal systems that impinge on LH-releasing hormone (LHRH)-synthesizing neurons. The reported absence of estrogen receptors (ERs) in LHRH neurons indicates that estrogen-receptive neurons that are afferent to LHRH neurons are involved in mediating the effects of this steroid. We now present evidence indicating that central histaminergic neurons, exclusively located in the tuberomammillary complex of the caudal diencephalon, serve as an important relay in this system. Evaluation of this system revealed that 76% of histamine-synthesising neurons display ERalpha-immunoreactivity in their nucleus; furthermore histaminergic axons exhibit axo-dendritic and axo-somatic appositions onto LHRH neurons in both the rodent and the human brain. Our in vivo studies show that the intracerebroventricular administration of the histamine-1 (H1) receptor antagonist, mepyramine, but not the H2 receptor antagonist, ranitidine, can block the LH surge in ovariectomized estrogen-treated rats. These data are consistent with the hypothesis that the positive feedback effect of estrogen in the induction of the LH surge involves estrogen-receptive histamine-containing neurons in the tuberomammillary nucleus that relay the steroid signal to LHRH neurons via H1 receptors.

Complement C5a anaphylatoxin fragment causes apoptosis in TGW neuroblastoma cells.

Human neuroblastoma TGW cells express a C5a anaphylatoxin receptor-like molecule termed neuronal C5a receptor. A C5a-receptor fragment peptide (termed PR226-multiple antigenic peptide) can induce rapid apoptosis in TGW cells via neuronal C5a receptor-associated signal transduction pathways. In order to analyse role of activated complement system in neurodegeneration, TGW cells were exposed to an oligomer form of a C5a fragment (amino acids: 37-53) peptide termed PL37-multiple antigenic peptide. Upon treatment with PL37-multiple antigenic peptide, an increased nuclear c-fos expression was shown within 30 min. DNA fragmentation, a hallmark of apoptosis, was noted within 4 h. Extracellular administration of 100 nM PL37-multiple antigenic peptide evoked inward calcium current pulses. At higher doses (0.5 microM-1 microM), PL37-multiple antigenic peptide evoked higher current pulses, followed by an irreversible, high inward current. To exert its apoptotic effect, PL37-multiple antigenic peptide utilizes a pertussis toxin-sensitive signal transduction pathway associated with the neuronal C5a receptor. Activation of the complement system and therefore release of C5a has already been reported in Alzheimer's disease. In addition, the presence of the Kunitz-type proteinase inhibitors indicates an impaired protease function and a possible abnormal fragmentation of C5a anaphylatoxin. Our data suggest that neurons expressing neuronal C5a receptor are more vulnerable to the apoptosis associated with the neuronal C5a receptor and the possibility that abnormal activation of C5a receptor and C5a anaphylatoxin fragments might be involved in the pathogenesis of Alzheimer's disease.

Partial characterization of a low molecular weight phagocytosis inhibitory factor obtained from human erythrocyte membranes.

Phagocytosis Inhibitory Factor (PIF), a small (< 3000 D) molecule, was partially purified from human red blood cell membranes. This factor inhibits latex phagocytosis by monocytic cells. PIF is not toxic under the experimental conditions employed and the phagocytosis inhibitory activity is reversible since removal of this factor restores the phagocytic capability of cells. The phagocytic activity of murine macrophages was not affected by PIF.

MHC-specific graft-protective and delayed-type hypersensitivity (DTH) suppressive activity of a CD4+ CD8+, alpha beta T cell receptor (TCR) positive lymphoma isolated from a tolerant mouse.

Two lymphomas were found in, and isolated from A (H-2a) mice in which permanent transplantation tolerance was induced to CBA (H-2k) histocompatibility antigens by the neonatal injection of (CBAxA)F1 spleen cells. They proved to be of recipient origin and were transferable to syngeneic A mice, growing as disseminated lymphomas (L33 and L46) and killing the recipients rapidly. Analysis of the cell surface antigens disclosed that both lymphomas had an immature T cell phenotype [Thy-1+, CD5+, CD3low, TCR alpha beta low, CD4low, CD8high, heat-stable antigen (HSA) positive, and CD44-, MHC class II-, CD45R-, sIg-, Gr-1-, CD11b-]. Intraperitoneal (i.p.) injection of syngeneic A mice with viable L33 lymphoma cells resulted in a dose-dependent, significant prolongation of the mean survival times of "specific" CBA and MHC-identical B10.BR skin allografts as compared to the survival of appropriate grafts in non-lymphoma-bearing controls. The survival times of third party MHC-incompatible B10 (H-2b) and B10.D2 (H-2d) allografts were only slightly prolonged in A mice inoculated with L33 cells. The graft-protective effect was not abrogated if the proliferative capacity of the L33 cells was blocked by in vitro mitomycin C (MMC) pretreatment. Furthermore, the inoculation of L33 lymphoma into A mice significantly inhibited their DTH response to the sensitizing CBA histocompatibility antigens. In contrast, the L46 lymphoma had no effect on the survival of CBA allografts and the DTH reactivity. These data suggest that the CD4+CD8+TCR alpha beta + L33 T cell lymphoma originating from a neonatally tolerant mouse has a specific immunosuppressive effect on the in vivo reactivity of syngeneic mice to the tolerance-inducing (MHC class I) alloantigens.

Endothelin (ET)-1 induced mucosal damage in the rat small intestine: role of ET(A) receptors.

The role of endothelin (ET)-1 as a mediator of small intestinal mucosal perfusion failure and tissue damage was investigated in the rat using intravital fluorescence videomicroscopy. The effects of intravenous infusion of ET-1 (3 nmol/kg) on functional capillary density, mucosal thickness, and the degree of mucosal damage were evaluated. Administration of ET-1 caused pronounced mucosal injury with a significant reduction of mucosal thickness compared with vehicle-treated control animals. Concomitantly, villous functional capillary density was markedly reduced 30 and 90 min after the infusion of ET-1. ET(A) receptor blockade by pretreatment with BQ 610 or with the novel ET(A) receptor antagonist ETR-P1/FL peptide prevented ET-1 induced capillary perfusion failure and mucosal damage. In contrast, the ET(B) receptor antagonist IRL 1038 was not effective. These results indicate that, acting via the ET(A) receptor, elevated levels of circulating ET-1 under various pathophysiological conditions, such as septic or hemorrhagic shock, might impair nutritive perfusion of the intestinal mucosa and contribute to tissue injury.

C5a receptor expression by TGW neuroblastoma cells.

We recently reported that not only lymphoid cells, but cells of neuronal origin may harbor C5a receptors (C5aR) as suggested by results of RT-PCR testing and that an apoptotic pathway is associated with the C5aR. To determine whether C5aR is expressed as an integral membrane protein, we generated mono- and polyclonal anti-C5aR antibodies. Flow cytometry showed a low-level expression of C5aR in TGW neuroblastoma cells. Epitope mapping suggested that a conformation change in C5aR occurs when exposed to C5a. Although an aphysiologically high concentration of C5a is necessary for inducing a transient increase in the intracellular Ca2+ level, TGW cells do employ the signal transduction pathway associated with C5aR, suggesting that these cells may serve as putative model for C5aR-expressing neurons.

Antisense homology box-derived peptides represent a new class of endothelin receptor inhibitors.

Several peptides encoded by the sense and corresponding antisense DNA have been found to recognize and bind to each other. We developed software to search for sense-antisense regions within proteins taking into account the degeneracy of the genetic code, i.e., one amino acid can have several "antisense" counterparts. Using this approach, we searched endothelin receptor type A for intramolecular regions related in sense-antisense fashion. After locating these regions (termed "antisense homology boxes"), several corresponding peptides were synthesized. The four new ET(A) receptor fragment peptides ETR-P1 ("CALSVDRYRAVASW"), ETR-P3 ("QGIGPLITAIEI"), ETR-P4 ("IADNAERYSANLSSHV") and ETR-P6 ("LNRRNGSLRIALSEHLKNRREVA") reported here can inhibit ET-1 activity.

Antisense homology boxes in C5a receptor and C5a anaphylatoxin: a new method for identification of potentially active peptides.

A computer program was designed to locate regions termed antisense homology boxes (AHB), i.e., 8-15 amino acid regions corresponding to complementary DNA strands. Two AHBs were found in C5a and eight within the C5a receptor. The majority of intermolecular AHBs were found to overlap those found in the intramolecular AHBs. Several AHB peptides were synthesized and tested for their ability to interfere with C5a receptor functions. A peptide fragment of the C5a receptor corresponding to the loop between the fifth and sixth hypothetical transmembrane regions (amino acids 226-245) antisense to C5a and an intramolecular AHB in C5a receptor proved to be an antagonist of C5a when preincubated with C5a at high concentrations (>0.5 microM). However, when U937 cells bearing the C5a receptor were preincubated with this peptide at a much lower concentration (even as little as 40 pmol), the AHB peptide behaved as an agonist. Another AHB peptide corresponding to region 10-27 in the C5a receptor bound to two of its corresponding antisense peptides derived from C5a anaphylatoxin, corresponding to amino acids 37-43 and 61-74. This observation raises the possibility that the C5a receptor may bind C5a with two distinct orientations. Two other AHB peptides derived from C5a, PL12 (amino acids 12-27), and PL61 (amino acids 61-74), were also shown to inhibit activity. Incubation of dibutyryl cyclic AMP-stimulated U937 cells with PL37 (amino acids 37-51) resulted in increased intracellular Ca2+ levels and an anergy to subsequent challenge with C5a. Locating regions with sense-antisense relationships in proteins might help in identification of peptides that can interfere with the function of target proteins.

Induction of transplantation tolerance and development of lymphomas in mice: lack of interdependence.

Neonatal transplantation tolerance was induced in five different H-2-incompatible donor----recipient mouse strain combinations by the iv injection of semiallogeneic (F1 hybrid) spleen cells. The highest degree of tolerance (expressed by the survival time of the specific test skin allografts) was observed in the (CBA X A)F1----CBA combination (approximately 95% permanent tolerance) but some degree of tolerance was achieved in all strain combinations. An increased incidence of malignant lymphoproliferative disorders was observed in all groups of mice which underwent neonatal tolerance induction. The highest incidence of lymphoproliferative malignancies was observed in the (B10 X A)F1----A tolerance induction system, in which approximately 50% of the recipient mice died within 1 year. In further experiments, spleen cells of mice which proved to be permanently tolerant after the neonatal tolerance induction were transferred into syngeneic, normal, adult, ATS-pretreated, allografted recipients; by this method, in approximately 50% of the recipients permanent "adult" tolerance was achieved. The spleen cells of the "adult" tolerant mice were able to transfer the tolerance to other adult, syngeneic, ATS-pretreated recipients. Even the fourth serial transfer resulted in essentially the same degree of tolerance in the new recipients. We consider the serial transfer a classical instance of "infectious tolerance" based on suppressor mechanisms. However, an increasing number (and malignancy) of lymphomas occurred in the course of the serial transfers and prevented the "indefinite" transfer of tolerance after the fourth occasion. We conclude that both the degree of transplantation tolerance and the high frequency of lymphomas are determined by (immuno)genetic factors but the two phenomena are not interrelated. Thus, successful transplantations do not seem to be necessarily accompanied by an increased incidence of malignancies.

Lethal systemic graft-vs-host disease in neonatally tolerant, but not in F1 hybrid mice.

The possibility, conditions, and quantitative aspects of eliciting GVHD in CBA (H-2k) mice made neonatally tolerant to the alloantigens of the donor A (H-2a) strain were studied. The intravenous injection of different doses (10(7)-2 x 10(8)) of A spleen cells caused a severe, often fatal, systemic GVHD in 12-month-old tolerant mice. The GVHD was found to be specific: spleen cells of a third party strain (B10) did not induce any disease. The intensity and the mortality of the GVHD depended on the cell dose and on the age of the recipients. In contrast, unirradiated (CBA x A)F1 recipients proved to be resistant to the lethal disease. In spite of their different susceptibility to the systemic GVHD, the tolerant and F1 hybrid recipients showed equally strong local GVH reactivity in the popliteal lymph node enlargement assay. Neonatally tolerant mice offer a new, sensitive model for the induction of lethal GVHD without the need of immunosuppression or irradiation.

Antisense sequences of 20-kDa homologous restriction factor (HRF20) are found in C9 and the C8 beta chain of homologous complement.

We examined, on the basis of the fact that sense and antisense peptides have affinity for each other, whether any relationship exists between homologous restriction factor (HRF20), a membrane inhibitor of the terminal stage of homologous complement attack, and the antisense sequence of the terminal complement components C8 and C9. In this article, we demonstrate that there are two regions of C9 that contain antisense sequences to one continuous region of HRF20 and that this relationship exists between human HRF20 and human C9, but not mouse C9. We also found one region of the C8 beta chain that contains an antisense sequence to HRF20.

Heterogeneous nuclear ribonucleoprotein D0 contains transactivator and DNA-binding domains.

Heterogeneous nuclear ribonucleoprotein D0 (hnRNP D0) is an abundant, ubiquitous protein that binds RNA and DNA sequences specifically, and has been implicated in the transcriptional regulation of the human complement receptor 2 gene. We found that in vivo expression of hnRNP D0-GAL4 fusion proteins increased the transcriptional activity of a GAL4-driven reporter gene, providing direct proof that hnRNP D0 possesses a transactivator domain. We found, using truncated hnRNP D0 proteins fused to GAL4, that 29 amino acids in the N-terminal region are critical for transactivation. We established, using a series of recombinant truncated hnRNP D0 proteins, that the tandem RNA-binding domains alone were not able to bind double-stranded DNA. Nevertheless, 24 additional amino acids of the C-terminus imparted sequence-specific DNA binding. Experiments using peptide-specific antisera supported the importance of the 24-amino-acid region in DNA binding, and suggested the involvement of the 19-amino-acid alternative insert which is present in isoforms B and D. The N-terminus had an inhibitory effect on binding of hnRNP D0 to single-stranded, but not to double-stranded, DNA. Although both recombinant hnRNP D0B and D0D bound DNA, only the B isoform recognized DNA in vivo. We propose that the B isoform of hnRNP D0 functions in the nucleus as a DNA-binding transactivator and has distinct transactivator and DNA-binding domains.

Cell-surface bound complement regulatory activity is necessary for the in vivo survival of KDH-8 rat hepatoma.

The monoclonal antibody 5I2 recognizes and functionally inhibits a Crry-like complement regulator molecule on rat cells (corresponding to human decay accelerating factor and membrane cofactor protein activity). The inhibition of complement regulatory activity by 5I2 antibody results in complement deposition on rat cell membranes exposed to homologous complement. Two subclones of KDH-8 rat hepatoma were selected for the experiments; one expressing high (CrryH) and the second low (CrryL) amounts of Crry-like antigen. Both sublines grow in vivo in syngeneic rats, but at lower cell doses (< 10(5) cells/rat) the survival time of rats injected with CrryL cells is prolonged. Injection of tumour-bearing rats with 5I2 monoclonal antibody intraperitoneally did not influence the tumour growth. However, it resulted in 50% mortality within a few hours, accompanied by severe haemorrhage in the peritoneal cavity. Pretreatment of the tumour cells with 5I2 monoclonal antibody or its F(ab')2 fragment substantially increased the survival time of recipient rats. Even permanent survivors were found, indicating that cell membrane-associated complement regulatory activity, which provides protection against attack of homologous complement, is necessary for in vivo tumour growth.

A low molecular weight phagocytosis-inhibitory factor obtained from human erythrocyte membranes specifically down-regulates Mac-1 activity on tetradecanoyl phorbol acetate-stimulated monocytic cell lines in a Ca(2+)-dependent manner.

A low molecular mass (< 3 kDa) phagocytosis-inhibitory factor (PIF), was partially purified from human erythrocyte membranes. PIF inhibits latex phagocytosis and C, as well as FcR-mediated phagocytosis, by macrophage-like cells in a Ca(2+)-dependent manner. This phagocytosis-inhibitory activity is reversible because removal of PIF restores phagocytic capability of cells. After treatment with PIF, Mac-1 Ag (CR3 or CD11b) becomes almost undetectable on the cell surface by immunofluorescence staining using the mAbs D-12 and BEAR-1, whereas staining with the LM2/1 anti-Mac-1 mAb proved that Mac-1 is still present on the cell surface, thus, indicating a possible conformational change in Mac-1. PIF has no significant inhibitory effect on staining of CR1 (CD35), CR2, (CD21), 20-kDa homologous restriction factor (CD59), decay-accelerating factor (CD55), LFA-1 (CD11a), or p150.95 (CD11c). Although binding of Mac-1-bearing U-937 cells to C3bi-opsonized beads is completely blocked, binding via Con A and FcRs remains unaffected by PIF treatment. In addition to the suppressive effect on phagocytosis, inhibition of cell adhesion was observed as well. The inhibitory effect of PIF on cell adhesion is not monocyte specific, because after exposure to PIF the TGW neuroblastoma cell line lost its ability to attach to the tissue culture plate, but retained its ability for homotypic aggregation. The possibility that PIF is a natural regulator of erythrophagocytosis is suggested.

Membrane-bound complement regulatory activity is decreased on vaccinia virus-infected cells.

Decay accelerating factor (DAF), membrane cofactor protein (MCP), complement receptor 1 and mouse Crry are cell surface-bound complement regulatory proteins capable of inhibiting C3 convertase activity on cell membranes, and therefore provide a substantial protection from attack by homologous complement activated either by the classical or by the alternative pathway. Decrease in complement regulatory activity might lead to spontaneous complement deposition and subsequent cell injury. MoAb 5I2 can inhibit the complement regulatory activity of molecules on rat cells, resulting in deposition of homologous complement. The antigen recognized by 5I2 MoAb in rats is homologous to mouse Crry. Fifteen to 20 h after infection with vaccinia virus, in vitro cultured KDH-8 rat hepatoma cells show a strong decrease in expression of Crry-like antigen, and proved to be sensitive to complement deposition when 1:5 diluted normal rat serum was added to the culture medium as a source of complement. Addition of complement to the cultured KDH-8 cells infected with a very low dose of vaccinia virus (1 plaque-forming unit (PFU)/1000 cells) substantially reduced spreading of virus infection in the cell culture, while inactivation of complement by heat or zymosan treatment abrogated the protective effect.

Antagonistic peptides against human anaphylatoxin C5a.

Multivalent synthetic peptides derived from C5a were prepared in order to examine their effects on the C5a receptor (C5aR). Multiple antigen peptide (MAP) of the C5a C-terminal region (MAP61-74) bound to cells expressing C5aR with high affinity. On the other hand, N-terminal peptides (MAP3-16 and MAP12-26) and one with a sequence from the mid-portion of C5a (MAP37-53) did not bind to the cells. In addition, MAP61-74 inhibited Ca2+ mobilization and release of beta-hexosaminidase by C5a from dibutyryl cAMP-activated U937 cells. This Ca2+ mobilization was also inhibited by MAP12-26 and Mono61-74, the monomeric C-terminal peptide. Taken together, these data indicate that C5a binds to the C5aR via its C-terminal region. Furthermore, MAP61-74, a 14mer peptide that has additional amino acids at the N-terminal compared with the C-terminal octapaptide, can bind to C5aR and can be considered an antagonist of C5a which may prove useful as an agent for controlling the allergic response caused by complement activation.