Molecular cloning, characterization of porcine IZUMO1, an IgSF family member.

IZUMO1, belonging to the family of mammalian immunoglobulin proteins, has been well characterized in the mouse. Here, we describe the molecular cloning and expression analysis of porcine IZUMO1 (pIZUMO1). Partial sequence information published in the National Center for Biotechnology Information (NCBI) database was used to generate the full-length sequence for IZUMO1 using rapid amplification of cDNA ends (RACE). A search of the porcine genomic sequence in the NCBI database identified a bacterial artificial chromosome (BAC) encoding the pIZUMO1 gene. This BAC is derived from porcine chromosome 6 and is syntenic with the corresponding regions of mouse, bovine, and human genomes encoding the IZUMO gene family. This BAC was found to encode an IZUMO1 protein with a predicted amino acid sequence having high similarity with mouse and human IZUMO1. Western blot analysis of proteins from porcine tissues indicated that pIZUMO1 was specifically expressed in the sperm. Furthermore, to confirm whether pIZUMO1 forms complexes, we overexpressed pIZUMO1 in HEK293 cells. The recombinant pIZUMO1 from cell extracts was found to form complexes. Our finding suggests that pIZUMO1 forms homodimeric complex on the sperm membrane. Furthermore, an IVF inhibition assay with an antibody for the porcine IZUMO1 Ig-like domain showed that Ig-like domain effectively prevented pig sperm-egg interactions.

Hydrodynamics-based gene delivery of naked DNA encoding fetal liver kinase-1 gene effectively suppresses the growth of pre-existing tumors.

Antiangiogenic gene therapy is a promising strategy for cancer treatment, which generally requires highly efficient delivery systems. To date, success of this strategy has depended almost exclusively on the delivery of high titers of viral vectors, which can result in effective transgene expression. However, their cytotoxicity and immunogenicity are a major concern for clinical applications. Recent advances in delivery efficiency of naked DNA could potentially meet the requirement for both high transgene expression and minimal side effects. To investigate whether naked DNA can be used for antiangiogenic cancer therapy, an expression plasmid was generated that encodes a soluble form of fetal liver kinase-1 (Flk-1) gene, a receptor for vascular endothelial growth factor (VEGF). Hydrodynamic injection of this plasmid resulted in close to 0.1 mg/ml of soluble Flk-1 protein in mouse serum and blocked VEGF-driven angiogenesis in matrigel in vivo. The same delivery significantly suppressed the growth of two different pre-existing subcutaneous tumors, Renca renal cell carcinoma and 3LL lung carcinoma. CD31 immunohistochemistry revealed that the tumor-associated angiogenesis was also highly attenuated in soluble Flk-1-treated mice. Thus, expression of genes by hydrodynamics-based gene delivery of naked DNA appears to be a promising approach for antiangiogenic cancer gene therapy.

Beta amyloid peptide (Abeta42) is internalized via the G-protein-coupled receptor FPRL1 and forms fibrillar aggregates in macrophages.

The 42 amino acid form of beta amyloid (Abeta42) plays a pivotal role in neurotoxicity and the activation of mononuclear phagocytes in Alzheimer's disease (AD). Our recent study revealed that FPRL1, a G-protein-coupled receptor, mediates the chemotactic and activating effect of Abeta42 on mononuclear phagocytes (monocytes and microglia), suggesting that FPRL1 may be involved in the proinflammatory responses in AD. We investigated the role of FPRL1 in cellular uptake and the subsequent fibrillar formation of Abeta42 by using fluorescence confocal microscopy. We found that upon incubation with macrophages or HEK293 cells genetically engineered to express FPRL1, Abeta42 associated with FPRL1 and the Abeta42/FPRL1 complexes were rapidly internalized into the cytoplasmic compartment. The maximal internalization of Abeta42/FPRL1 complexes occurred by 30 min after incubation. Removal of free Abeta42 from culture supernatants at 30 min resulted in a progressive recycling of FPRL1 to the cell surface and degradation of the internalized Abeta42. However, persistent exposure of the cells to Abeta42 over 24 h resulted in retention of Abeta42/FPRL1 complexes in the cytoplasmic compartment and the formation of Congo red positive fibrils in macrophages but not in HEK 293 cell transfected with FPRL1. These results suggest that besides mediating the proinflammatory activity of Abeta42, FPRL1 is also involved in the internalization of Abeta42, which culminates in the formation of fibrils only in macrophages.

Development of a homogeneous time-resolved fluorescence assay for high throughput screening to identify Lck inhibitors: comparison with scintillation proximity assay and streptavidin-coated plate assay.

This study details the development of a homogeneous time-resolved fluorescence (HTRF) high throughput screening assay to identify inhibitors of Lck. HTRF was compared with scintillation proximity and streptavidin-coated plate assays. Because of the differences in the sensitivity of detection of phosphotyrosine among the three assays, different amounts of enzyme were used. However, the concentrations of the other assay components were standardized. When using similar assay conditions, the calculated IC(50) values of inhibitory compounds were independent of assay format. Furthermore, filtration experiments revealed that phosphorylation of a biotinyl poly-Glu,Ala, Tyr peptide substrate was less than autophosphorylation of the Lck enzyme; this was due to the low K(m) value for biotinyl poly-Glu,Ala,Tyr. In the HTRF assay, small amounts of enzyme and high concentrations of ATP could be used, thereby minimizing the effects of autophosphorylation. Higher ATP concentration would also minimize the effect of ATP competitors. Using this technology, it may be possible to find novel kinase inhibitors that do not act at the ATP binding site of protein tyrosine kinases.

Endothelin receptors and their cellular signal transduction mechanism in human cultured prostatic smooth muscle cells.

1. Endothelin (ET) receptors, and their cellular signal transduction mechanism, were characterized in a primary culture of human prostatic smooth muscle cells (HP cell). 2. [125I]-ET-1 and [125I]-ET-3 binding studies revealed that both ETA and ETB receptors were present in the HP cells, and the ratio of ETA to ETB receptors was 1.4:1. 3. Analysis of ET receptor mRNA by reverse transcription-polymerase chain reaction also demonstrated that HP cells express both ETA and ETB receptors. 4. ET-1 and ET-3 increased intracellular free Ca2+ concentration ([Ca2+]i) in the HP cells in a concentration-dependent manner. Use of subtype selective antagonists BQ-123 and BQ-788, indicated that both ETA and ETB receptors were coupled to an increase in [Ca2+]i. 5. Pretreatment of the cells with pertussis toxin resulted in a significant but partial attenuation of the [Ca2+]i increase mediated through the ETA and ETB receptors. However, sensitivity to pertussis toxin (PTX) was significantly different between them. 6. In conclusion, HP cells possess ETA and ETB receptors. Further, these two endothelin receptor subtypes evoke an increase in [Ca2+]i possibly via the action of different GTP-binding proteins.

Oxytocin receptors expressed and coupled to Ca2+ signalling in a human vascular smooth muscle cell line.

1. In a human vascular smooth muscle cell line (HVSMC), binding experiments with [3H]-arginine8-vasopressin (AVP) have shown the existence of a homogeneous population of binding sites with affinity (Kd value) of 0.65 nM and a maximum number of binding sites (Bmax) of 122 fmol mg-1 protein. 2. Nonlabelled compounds compete for [3H]-AVP binding in the HVSMC membrane with an order of potency of oxytocin > lyspressin > or = AVP > Thr4, Gly7-oxytocin > (beta-mercapto-beta-beta-cyclopentamethylenepropionyl-O-Me Tyr2, Arg8) vasopressin > desmopressin > OPC21268 > OPC31260. This order was markedly different from that observed in rat vascular smooth muscle cells (A10), a well-established V1A receptor system. 3. In HVSMC both oxytocin and AVP increased inositol 1,4,5-trisphosphate (IP3) production and [Ca2+]i response, but the efficacy of the responses was greater for oxytocin than AVP. 4. Reverse transcription-polymerase chain reaction (RT-PCR) assay detected only oxytocin receptor but not V1A or V2 receptors in HVSMC, whereas only V1A receptors were found in A10 cells. 5. In conclusion, in HVSMC only oxytocin receptors are expressed among the vasopressin receptor family, and they coupled to phosphatidyl inositol (PI) turnover/Ca2+ signalling. This unexpected observation should provide new insight into the functional role of the oxytocin receptor in a human vascular smooth muscle cell line.

Characterization of YM022: its CCKB/gastrin receptor binding profile and antagonism to CCK-8-induced Ca2+ mobilization.

We investigated the antagonistic activity of (R)-1-[2,3-dihydro-1-(2'- methylphenacyl)-2-oxo-5-phenyl-1H-1,4-benzodiazepin-3-yl]-3- (3-methylphenyl) urea (YM022), a benzodiazepine derivative, at CCKB/gastrin receptors. This compound potently inhibited [125I]CCK-8 binding to rat brain CCKB/gastrin receptors with a Ki value of 0.26 nM, but it showed weak affinity for rat pancreas CCKA receptors (Ki = 270 nM). Selectivity for CCKB/gastrin receptors was 1000-fold greater than that for CCKA receptors. Changes in intracellular free Ca2+ concentration ([Ca2+]i) in response to CCK-8 were measured in a rat anterior pituitary cell line GH3 by fura-2 fluorometry. CCK-8 (1-100 nM) dose-dependently increased [Ca2+]i in these cells, whereas YM022 had no effect on baseline [Ca2+]i even at the highest concentration of 100 nM. YM022 inhibited the mobilization of [Ca2+]i elicited by 10 nM CCK-8 in a concentration-dependent manner with an IC50 value of 4 nM. In conclusion, YM022 is an extremely potent and highly selective antagonist of CCKB/gastrin receptors. This compound is therefore useful for studying the physiological and pharmacological roles of CCKB/gastrin receptors.

Mitogenic activity of endothelin on human cultured prostatic smooth muscle cells.

The effects of endothelins on human prostatic smooth-muscle cell growth were examined. Endothelin-1 and endothelin-3 induced a concentration-dependent increase in DNA synthesis and also promoted cell growth. Use of subtype selective antagonists BQ-123 ((cyclo(D-Trp-D-Asp(ONa)-Pro-D-Val-Leu); endothelin ET(A) receptor selective) and BQ-788 ((N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methyl Leu-D-Trp-(COOMe)-D-Nle-ONa); endothelin ET(B) receptor selective), indicated that mitogenic effects of endothelin were mediated through activation of both endothelin ET(A) and ET(B) receptors. The mitogenic effects of endothelin-1 and endothelin-3 were significantly inhibited by pretreatment of the cells with pertussis toxin. However, mitogenesis due to basic fibroblast growth factor was not affected. In conclusion, endothelin has mitogenic effects on human prostatic smooth muscle cells through activation of both endothelin ET(A) and ET(B) receptors via different signalling pathways from basic fibroblast growth factor. This may contribute to smooth muscle hyperplasia associated with benign prostatic hyperplasia.

Glucocorticoid hormone can suppress apoptosis of rat testicular germ cells induced by testicular ischemia.

OBJECTIVE: To evaluate the effect of dexamethasone, a potent synthetic glucocorticoid hormone, on apoptosis of testicular germ cells and vascular neutrophil adhesion after repair of testicular torsion in rats. DESIGN: Controlled laboratory study. SETTING: Department of Urology, Yamagata University School of Medicine, Yamagata, Japan. ANIMAL(S): Fifty-one male Sprague-Dawley rats. INTERVENTION(S): Dexamethasone, 10 mg/kg of body weight. MAIN OUTCOME MEASURE(S): Testicular germ-cell apoptosis (percentages of apoptotic tubules and apoptotic cells) and vascular neutrophil adhesion were assessed by using DNA nick-end labeling and the endothelial-neutrophil adhesion score, respectively. RESULT(S): Intravenous administration of dexamethasone at repair of 90-minute testicular torsion significantly inhibited testicular germ-cell apoptosis and vascular neutrophil adhesion. This inhibition was suppressed by intravenous injection of mifepristone, a glucocorticoid-receptor antagonist. CONCLUSION(S): Glucocorticoids can be administered for torsion in addition to conventional torsion repair.

Intracytoplasmic sperm injection using immobilized or motile human spermatozoon.

OBJECTIVES: To investigate the efficacy of the treatments for oocyte activation on the results of intracytoplasmic sperm injection using immobilized or motile human spermatozoa. DESIGN: The protocol of intracytoplasmic sperm injection was divided into four groups according to the states of sperm used for microinjection and the treatment for oocyte activation. In group A, immobilized sperm is used. The oocyte is activated merely by aspiration of the cytoplasm into the pipette. In group B, immobilized sperm is used. Microinjected oocyte is treated with A23187. In group C, immobilized sperm is used. Electroporation is performed on the microinjected oocyte. In group D, motile sperm is used. The oocyte is activated merely by aspiration of the cytoplasm into the pipette. SETTING: The Obstetrics and Gynecology Hospital, Fukushima Medical College. PATIENTS: The subjects are the cases that had failed fertilization in standard IVF, cases of severe oligozoospermia, and cases of severe asthenozoospermia. RESULTS: No difference was found between the groups as to the survival rate and fertilizing rates of oocytes after intracytoplasmic sperm injection. The cleavage rate of oocytes was high in order of group D, C, B, A. The cleavage rate for groups D, C, and B was significantly higher than group A. Cases of pregnancy were found in groups D and B. CONCLUSION: Using motile sperm rather than immobilized sperm can be expected to produce better results in human ICSI. Activating oocytes positively is needed when immobilized sperm is used.

Fertilization using male germ line-cells.

In recent years, favorable results have been achieved in patients suffering from azoospermia by microinsemination of spermatozoa taken from their testes. Microinsemination is being introduced in the treatment of patients who have no spermatozoa in their testes via their spermatid and spermatocyte. There are still doubts relating to immature male germ line-cells, such as whether they have, oocyte activating factors, the level of stability of DNA of cell nuclei, and the differences in chromosome numbers. The relatively few cases of gestation using the human spermatid treatment may be due to embryological problems resulting from the instability of nuclear DNA and the insufficiency of oocyte activating factors, which are the result of imperfect microinjection techniques. Improvements in techniques for the clinical application of spermatid and secondary spermatocyte, as well as the collection of basic data to confirm embryological safety are therefore necessary.

Studies on tomaymycin. III. Synthesis and antitumor activity of tomaymycin analogs.

The syntheses and antitumor activity of tomaymycin analogs are described. Structural modification of such parts of tomaymycin as the ethylidenepyrrole ring, animal bond, and substituents of the benzene ring are discussed.

Reduction of serum interleukin-5 levels reflect clinical improvement in patients with atopic dermatitis.

Cytokines, in particular IL-4 and IL-5, regulate IgE synthesis and eosinophil activation in atopic dermatitis (AD). To elucidate whether the serum levels of IL-4 and IL-5 are related to the serum IgE level, eosinophilia, or clinical severity of the disease, 25 cases with AD were studied. Blood samples were isolated from two groups of donors: 1) patients with AD (n = 25); 2) non-allergic individuals (NA, n = 20) with serum IgE levels below 100 IU/ml and with blood eosinophil counts below 250/microliter. Each parameter was evaluated at least twice in AD patients at the beginning of the study and after 4, 8 or 12 weeks of treatment. IL-4 was hardly detected in AD and NA, but IL-5 was increased (> 10 pg/ml) in most cases (22/25) of AD group with 513.6 pg/ml as the mean. AD with normal serum IgE levels exhibited increased levels of IL-5, whereas AD with high serum IgE levels did not necessarily have elevated IL-5 levels. The IL-5 level tended to change in parallel with the clinical severity in each AD case, although the level itself was not correlated with the clinical severity per se. A significant decrease of IL-5 was observed in AD when the clinical severity decreased. Eosinophils also decreased along with the improvement of AD, whereas the serum level of IgE did not change during the observation period. Our results suggest that IL-5 is involved in the regulation of clinical courses of AD and that its kinetics at the serum level reflects the clinical activity of AD.

Successful treatment of a patient with lymphomatoid papulosis by methotrexate.

We report a case of lymphomatoid papulosis (LyP) that occurred in a 44-year-old Japanese male patient. Reddish papules with a small number of pustules and nodules were observed on the extremities, chest and upper back. Most lesions were also associated with central necrosis, ulceration and crusting, and regressed spontaneously within 4 to 6 weeks. Histopathological examination revealed wedge-shaped dense cellular infiltrate in the dermis, which was mixed with large atypical lymphoid cells, small lymphocytes, eosinophils and neutrophils. These large atypical cells expressed CD30 on their cell membrane and cytoplasm. Rearrangement of the T-cell receptor (TcR) beta-chain gene was detected in the skin lesion. Lymphadenopathy with histopathologic change similar to the skin lesions, but without TcR gene rearrangement, was found at the left inguinal area. Systemic administration of methotrexate (7.5-15.0 mg/week) was found to be dramatically effective in resolution of skin lesions and prevention of their recurrence.

Morphometrical observations on the mandible of five strains of rabbits and strain identification using mandible measurements.

In order to clarify the relationships between five strains of rabbits and to identify the strains, principal component and discriminant analyses were carried out using 12 mandibular measurements of three inbred strains (JW-NIBS/Y (JW/Y), NW-NIBS/Y (NW/Y) and Dutch-NIBS/Y (D/Y)) and two outbred strains (JW-NIBS (JW) and NW-NIBS (NW] which were maintained at the Nippon Institute for Biological Science. The results obtained were as follows. (1) Principal component analysis revealed that in the males the mandible of JW was the largest but with considerable variation. D/Y was the smallest of all strains examined. The mandibles of NW and NW/Y were similar to the JW mandibles but had a shape which was shorter and higher. In the females the mandible of NW was the largest of all strains and, as in the males, D/Y was the smallest and JW varied markedly. (2) Discriminant analysis showed the probability of erroneous discrimination to be 14.8% (34/229) when the inbred and outbred strains were combined. In both sexes erroneous discrimination mostly occurred between NW and NW/Y, which have the same origin, and between JW and NW, which have a common ancestor. However, when the inbreds and outbreds were identified separately by discriminant analysis, the probability of erroneous discrimination was low in both cases (4.5% (5/112) and 7.7% (9/117) respectively). These results indicate that strain differences are found in the size and shape of rabbit mandibles and that mandible analysis is effective for strain identification of laboratory rabbits.

Hyperbaric oxygenation therapy for cyclophosphamide-induced haemorrhagic cystitis.

A 41-year-old female patient undergoing cyclophosphamide therapy for ovarian tumour suffered from haemorrhagic cystitis. Hyperbaric oxygenation (HBO) (100% of oxygen concentration at 2 atmospheric absolute pressures for 60 minutes a day for 30 days) was performed. After treatment the symptom subsided and haematuria disappeared. Cystoscopic findings demonstrated marked improvement. During the course of therapy no side effect was noted. Thus, HBO treatment appeared to have a beneficial effect on cyclophosphamide cystitis.

[A study of relation between phrasing and intertrial interval in reinforcement pattern learning in rats].

Effects of the temporal interval on reinforcement pattern learning were investigated in four experiments using a runway. The reinforced trial was always the first trial for R5N sequence (Experiments 1a and 1b), and the fourth trial for 3NR2N sequence (Experiments 2a and 2b). Group S-ITI received the given sequence at 30-s ITI, Group L-INT received the same procedure as Group S-ITI except for a 30-min ITI inserted between Trials 3 and 4 (Experiments 1a and 2a). Group L-ITI received at 30-min ITI, Group S-INT did as Group L-ITI except for the ITI between Trials 3 and 4 was 30 seconds (Experiments 1b and 2b). It was found that the running speed on each of Trials 1 and 4 was faster than any other trials under R5N and 3NR2N sequences for Group L-INT. That is, the running pattern for Trials 1-3 was similar to that for Trials 4-6. On the other hand, the running speed on Trial 4 under R5N sequence and that on Trial 1 under 3NR2N sequence did not increase for Group S-INT. These results suggest that a longer or shorter ITI plays the roles of both phrasing cue and discriminative stimulus. For Group S-ITI and Group L-ITI, there was little evidence that sequences were phrased. Therefore, when trials are separated by equal ITIs, neither 30-s ITI nor 30-min ITI becomes a phrasing cue.

[Short intertrial interval as a phrasing cue in the reinforcement-pattern learning in rats].

The effect of short intertrial interval as a phrasing cue on the reinforcement-pattern learning was investigated in a rat experiment using a runway. In the acquisition session, 20 rats were trained for NNR sequence in which two nonreinforced trials were followed by a reinforced one with equal ITI of 30 min. In the sequence-addition session, a new sequence with consisting of three nonreinforced trials with 30-min ITI was added 30 min and 30 s after the original sequence of trials for Group L-ITI and for Group S-INT, respectively. Rats of Group S-INT responded on trials 4-6 with the similar pattern of running speed as they showed on Trials 1-3 from the second day. In Group L-ITI, the running speed on Trial 4 did not decrease. This suggests that Group S-INT rats learn to use the ITI of 30 s as a phrasing cue not on the first day but from the second day on. The phrasing effect of short ITI (30 s) was discussed in comparison with that of longer ITI (30 min).

[Short intertrial interval as a phrasing cue in the serial pattern learning in rats].

Phrasing effect of short intertrial interval (ITI) on the serial pattern learning was investigated in a runway experiment. In acquisition, all 10 rats were given three repetitions of 14-7-3-1-0 subpattern daily over Days 1-80. For Group NP (non phrasing), all daily 15 trials were separated by a 15-min ITI. Group GP (good phrasing) and Group BP (bad phrasing) received the same procedure as Group NP except, instead of the 15-min ITI, a 15-s ITI was inserted between the 0 pellet and 14 pellets trial in the former, and a 15-s ITI was inserted between the 3 pellets and 1 pellet trial in the latter. Means of the last 4 days of acquisition as indicated by running speed showed that Group GP produced better anticipation of the 0 pellet trial than Group NP, and that Group BP produced the poorest anticipation. In Group BP, rats ran more slowly on the 1 pellet trial, that occurred after the 15-s ITI, than on the 0 pellet trial in each of the first and second subpatterns. These results suggest that a shorter ITI plays dual roles, as a phrasing cue and as a discriminative stimulus in the serial pattern learning as that does in reinforcement-nonreinforcement pattern learning.

[Interval effects of added sequences on reinforcement pattern learning in rats].

Three experiments examined how intervals of added sequences affected rat's learning of the reinforcement pattern. Animals were trained for runway performance corresponding to the series of one reinforcement trial (R) and two nonreinforcement trials (N) run with 30-s ITIs. The series was NNR, RNN, NRN for Experiments 1, 2, 3, respectively. Following this acquisition training, a second series of three nonreinforced trials with 30-s ITIs was added to the first series. Animals were assigned to two groups matched for performance level and they were given an added series with either long or short inter-session intervals. Subjects in Group S-ITI were given totally six trials with 30-s ITIs, while subjects in Group L-INT were given 30-min between the first and second series. Running speed for the first series differed with structure of the series (reinforcement pattern). The pattern of running speed for added series (Trials 4-6) was similar to that for original series (Trials 1-3) in Group L-INT, while running speed was kept at a low level for added series of nonreinforced trials in Group S-ITI. As is suggested by these findings, the events regularly occurred with equal ITIs can be remembered as one series, even when a new series of events is added to already experienced one with the same ITIs. However, when the second series is temporally separated from the first one by long intervals, the memory system may be reset so that events can be segregated as two sequences.