Lenalidomide induces cell death in an MDS-derived cell line with deletion of chromosome 5q by inhibition of cytokinesis.

Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders characterized by refractory cytopenias and susceptibility to leukemic transformation. On a subset of MDS patients with deletion of the long arm of chromosome 5 (del(5q)), lenalidomide exerts hematological and cytogenetic effects, but the underlying pharmacological mechanisms are not fully understood. In this study, we have investigated the in vitro effects of lenalidomide on an MDS-derived cell line, MDS-L, which carries del(5q) and complex chromosome abnormalities. We found that the growth of MDS-L cells was specifically suppressed mainly by apoptosis, and in addition, multinucleated cells were frequently formed and finally died out in the presence of lenalidomide. Time-lapse microscopic observation and the DNA ploidy analysis revealed that lenalidomide does not affect DNA synthesis but inhibits cytokinesis of MDS-L cells. The gene expression profile showed decreased expression of M phase-related genes such as non-muscle myosin heavy-chain 10, polo-like kinase 1, aurora kinase B, citron kinase and kinesin family member 20A(KIF20A). Interestingly, KIF20A is located at 5q31. These data contribute to the understanding of action mechanisms of lenalidomide on MDS with del(5q) and complex abnormalities.

Effect of sample collection site on semen parameters of healthy young volunteers.

The effect of sample collection site on semen parameters in ten men aged between 22 and 24 years was investigated. Sperm was collected at two sites: in a university hospital restroom for general use and in a one-person hospital room. Samples were collected from the same individual twice, with an interval of two weeks between collections. Semen parameters for the two sites were compared. Samples were collected after a minimum of three days and not longer than seven days of sexual abstinence. Sperm concentration did not differ significantly between the university hospital restroom location (86.8 +/- 25.4 x 10(6)/ml; mean +/- standard deviation) and the private hospital room (97.1 +/- 72.0 x 10(6)/ml). There was no difference in the total motile sperm count or daily sperm production between the collection sites. These results suggest that the collection site has little effect on semen parameters.

Effects of pentadecapeptide BPC157 on regional serotonin synthesis in the rat brain: alpha-methyl-L-tryptophan autoradiographic measurements.

A novel pentadecapeptide, BPC157, was recently reported to have a large spectrum of in vivo activities, from anti-ulcer to central action on the brain dopaminergic system. The mechanisms of these actions are not well understood. In this study, the evaluation of the effects of acute and repeated administration of BPC157 on serotonin (5-HT) synthesis in the rat brain is reported. The alpha-[14C]methyl-L-tryptophan (alpha-MTrp) autoradiographic method was used to measure regional 5-HT synthesis rates. In the first series of experiments, a single dose treatment of BPC157 (10 microg/kg) administered intraperitoneally 40 min before the alpha-MTrp tracer injection significantly reduced the regional rate of 5-HT synthesis in the dorsal thalamus, hippocampus, lateral geniculate body and hypothalamus. 5-HT synthesis rates in the substantia nigra reticulate and medial anterior olfactory nucleus in BPC157 treated rats were significantly higher than in the control rats. No significant change in the synthesis rate was observed in the raphe nuclei. In the second series of experiments, following a 7-day treatment with BPC157 (10 microg/kg; s.c.), a significant reduction in the 5-HT synthesis rate was observed in the dorsal raphe nucleus, and significant increases were observed in the substantia nigra, lateral caudate, accumbens nucleus and superior olive. This data suggests that BPC157, a gut peptide, influences brain 5-HT synthesis in rats, but we cannot determine, from this data, the mechanism of this action.

Effects of acute or chronic administration of anti-migraine drugs sumatriptan and zolmitriptan on serotonin synthesis in the rat brain.

Triptans are 5-HT1 receptor agonists used as anti-migraine drugs. They act primarily on meningeal blood vessels and on trigeminovascular afferents, but they may also exert central effects. We studied the regional effects of acute and chronic treatment with sumatriptan or zolmitriptan on the rate of serotonin (5-HT) synthesis in the rat brain, using the alpha-14C-methyl-L-tryptophan quantitative autoradiographic method. Sumatriptan at low (300 microg/kg, s.c.) and high (1 mg/kg) doses, as well as zolmitriptan (100 microg/kg), acutely decreased (15-40%, P < 0.05-0.001) 5-HT synthetic rate in many brain regions, including the dorsal raphe nucleus. Chronically, sumatriptan (21 days, approximately 300 microg/kg per day via osmotic minipumps) induced significant increases in the 5-HT synthesis rate in many projection areas but had no effect in the dorsal raphe nucleus. The acute effects on 5-HT synthesis rate would be compatible with activation of 5-HT1 autoreceptors that inhibit serotonin release. In contrast, the increased 5-HT synthesis rate observed after chronic sumatriptan might possibly result from a down-regulation/desensitization of 5-HT1 receptors and/or unmasking of excitatory triptan-sensitive 5-HT receptors. Overall, the present findings indicate that not only zolmitriptan but also sumatriptan affect brain serotonergic neurotransmission.

Individual variation in semen parameters of healthy young volunteers.

Individual variation in semen parameters was investigated in healthy young volunteers. Semen samples were collected approximately once a month over a one-year period for a total of 93 samples (5 to 10 samples per subject) from 12 volunteers in their twenties. Semen analysis was carried out according to the WHO Manual. The amount of variation in each semen variable was calculated for each subject by dividing the maximum value by the minimum value. The results showed that the semen volume varied by 1.9+/-0.8 fold (1.3 to 4.2 fold), the sperm concentration by 4.8+/-4.3 fold (1.5 to 17.2 fold), the percentage of sperm with forward progression by 2.8+/-1.4 fold (1.6 to 6.4 fold), the percentage of sperm with rapid linear progression by 3.4+/-2.6 fold (1.7 to 10.9 fold), the percentage of sperm with normal morphology by 1.9+/-0.4 fold (1.3 to 2.4 fold), and the percentage of live sperm by 1.5+/-0.4 fold (1.1 to 2.6 fold). A between-group comparison showed significant differences in all of the variables except the percentage of sperm with normal morphology. These results suggest multiple and considerable semen analyses are needed when evaluating semen parameters.

Diagnosis and treatment of progressive space-occupying radiation necrosis following stereotactic radiosurgery for brain metastasis: value of proton magnetic resonance spectroscopy.

BACKGROUND: There have been some reports that radiation necrosis can be controlled conservatively. There are rare cases showing progressive space-occupying radiation necrosis (PSORN). It is very difficult to control PSORN by conservative treatment. The purpose of this study was to evaluate the early diagnosis of those cases and the timing of surgery for patients with PSORN. METHOD: We have experienced some cases where quality of life was improved by the removal of PSORN after stereotactic radiosurgery (SRS) for brain metastases. Therefore, we evaluated retrospectively the diagnosis and treatment of six cases of symptomatic PSORN at approximately 6-12 months after SRS for metastatic brain tumours. FINDINGS: In all six cases, on Magnetic Resonance Imaging with Gd contrast material (Gd-MRI), PSORN was revealed as a ring-like enhanced mass with large perifocal oedema coupled with the appearance of neurological deficit. Proton Magnetic Resonance Spectroscopy ((1)H-MRS) enabled us to differentiate PSORN from recurrence of metastases in all six cases. Single Photon Emission Computed Tomography with thallium-201 chloride (201TlCl-SPECT) enabled us to do this in four cases of the six. In four cases of the six, lesionectomy of the ring-like enhanced mass (PSORN) was performed, and in two of these cases the removal was performed within 4 weeks from the time when conservative treatment became ineffective, and the neurological deficit and perifocal oedema was improved as was the quality of life. However, in the other two patients who were left for more than 16 weeks, the deficit was gradually progressive. The two patients who did not receive lesionectomy were treated by conservative means with steroids and/or heparin and warfarin and they had progressive neurological symptoms. INTERPRETATION: Although, the number of patients is small in this study, and more data will be needed, it is recommended that lesionectomy is performed at an early stage, if possible, when conservative management has failed.

Adhesion via CD43 induces Syk activation and cell proliferation in TF-1 cells.

The effect of adhesion via CD43 (leukosialin, sialophorin) on cell proliferation and phosphorylation signaling were examined in a growth factor-dependent hematopoietic progenitor cell line, TF-1. TF-1 cells promptly resulted in death after withdrawal of growth factors. However, the viable cell number increased when TF-1 cells were cultured on anti-CD43 monoclonal antibody-coated plates. In this case, sustained activation of protein tyrosine kinase Syk and extracellular signal-regulated kinase (Erk) 1/2 were detected. Overexpression of exogenous Syk on TF-1 cells by the adenovirus vector system induced enhancement of the cell proliferation accompanied with enhancement of the Erk activation by a dominant-positive effect. The signal transducer and activator of transcription (STAT) 5 seemed not to be associated with the CD43-mediated cell proliferation. These results indicated that adhesion via CD43 induces the proliferation of TF-1 cells in the absence of growth factors in part by Syk-dependent Erk 1/2 signaling.

Neurotrophin secretion from cultured microglia.

Because microglia have been suggested to produce neurotrophins, we tested this ability in vitro. Rat primary microglia were found to constitutively secrete a limited amount of brain-derived neurotrophic factor (BDNF), but nerve growth factor (NGF) and neurotrophin-3 (NT-3) were undetectable in the conditioned medium. Stimulation of the cells with lipopolysaccharide (LPS) increased BDNF secretion, and induced NGF secretion. As a first step to examine this regulation system, the association of protein kinase C (PKC) was pharmacologically analyzed. A PKC activator, phorbol-12-myristate-13-acetate, enhanced the secretion of BDNF. Pre-treatment of microglia with a PKC inhibitor, bisindolylmaleimide, suppressed LPS-stimulated BDNF secretion as well as the constitutive one. These results suggest that the PKC signaling cascade is closely associated with BDNF secretion. Among PKC isoforms, PKCalpha probably plays a role in BDNF secretion, based on the results of experiments using a specific PKC activator, 1-oleoyl-2-acetyl-sn-glycerol, and a specific PKC inhibitor, Gö 6976, and by immunoblotting. Taken together, these findings suggest that the secretion of BDNF from microglia is regulated through PKCalpha-associated signal transduction mechanism.

Ability of rat microglia to uptake extracellular glutamate.

Since it has been suggested that microglia in vivo act as glutamate scavengers, this possibility was investigated in primary cultured microglia. The microglia showed specific abilities to uptake (14)C-glutamate depending on incubation time and numbers of cells used. The activity was suppressed by a specific inhibitor for a glial cell-type transporter, glutamate transporter-1 (GLT-1) (EAAT2). However, that of cultured astrocytes was not affected. These results suggest that microglia uptake glutamate by means of GLT-1. Supporting these results, immunoblotting revealed the presence of GLT-1 in the microglia, while only glutamate-aspartate transporter (GLAST) (EAAT1: another glial cell-type transporter) was detected in the astrocytes. All together, these results indicate that microglia can act as glutamate scavengers in vivo by expressing the glutamate transporter GLT-1.

Effects of selective 5-HT1A receptor antagonists on regional serotonin synthesis in the rat brain: an autoradiographic study with alpha-[14C]methyl-L-tryptophan.

The effects of acute and chronic administration of WAY100635 and WAY100135, serotonin (5-HT)1A antagonists, on 5-HT synthesis rates, calculated from the trapping of alpha-[14C]methyl-L-tryptophan (alpha-MTrp), were evaluated in the rat brain using autoradiography. In the acute treatment studies, WAY100635 (1 mg/kg) induced a significant increase in 5-HT synthesis in the median raphe nucleus and some nerve terminal structures (range between 18 and 53%), while WAY100135 (10 mg/kg) produced a significant decrease of synthesis, in the range between 16 and 33%, in the raphe magnus nucleus and several projection areas. The action of WAY100635 given acutely was likely a result of antagonist actions at the 5-HT1A somato-dendritic autoreceptors. WAY100135 probably acted acutely as a partial agonist. In the chronic treatment studies, WAY100635 (1 mg/kg/day) and WAY100135 (10 mg/kg/day) were administered for 7 days as s.c. injections once a day. Chronic treatment with both compounds significantly reduced the rate of 5-HT synthesis in the nerve terminal structures and produced a significant increase in the raphe nuclei. These treatments did not have any effect on the plasma free or total tryptophan.

Caspase-3 activation by lysosomal enzymes in cytochrome c-independent apoptosis in myelodysplastic syndrome-derived cell line P39.

In most cases, apoptosis is considered to involve mitochondrial dysfunction with sequential release of cytochrome c from mitochondria, resulting in activation of caspase-3. However, we found that etoposide induced apoptosis in P39 cells, a myelodysplastic syndrome-derived cell line, without the release of cytochrome c. Furthermore, in etoposide-treated P39 cells, no changes in mitochondrial membrane potential (delta psi m) were detected by flow cytometry. Flow cytometry using a pH-sensitive probe demonstrated that lysosomal pH increased during early apoptosis in P39 cells treated with etoposide. A reduction in the ATP level preceded the elevation of lysosomal pH. In addition, specific inhibitors of vacuolar H+-ATPase induced apoptosis in P39 cells but not in HL60 cells. Although etoposide-induced activation of caspase-3 was followed by DNA ladder formation in P39 cells, E-64d, an inhibitor of lysosomal thiol proteases, specifically suppressed etoposide-induced activation of caspase-3. Western blotting analysis provided direct evidence for the involvement of a lysosomal enzyme, cathepsin L. These findings indicate that lysosomal dysfunction induced by a reduction in ATP results in leakage of lysosomal enzymes into the cytosolic compartment and that lysosomal enzyme(s) may be involved in activation of caspase-3 during apoptosis in P39 cells treated with etoposide.

Ceramide-enhanced urokinase-type plasminogen activator (uPA) release is mediated by protein kinase C in cultured microglia.

As described previously, a relatively high dose of neurotrophins increased the release of urokinase-type plasminogen activator (uPA) from cultured microglia. This biological response is suggested to be caused by ceramide, which is a metabolite of nerve growth factor low-affinity receptor (NGFRp75)-associated sphingomyelin turnover. Therefore, in the present study, we examined the effect of ceramide on the release of uPA from cultured microglia. Treatment of the cells with permeable C8-ceramide (D-erythro-Sphingosine, N-octanoyl-) enhanced uPA release in a dose-dependent manner. This effect of C8-ceramide was mimicked by treatment with bacterial sphingomyelinase. A pharmacological study using a specific PKC activator, phorbol-12-myristate-13-acetate, and a protein kinase C (PKC) inhibitor, bisindolylmaleimide, showed that PKC activation is required in order to release uPA from ceramide-stimulated microglia as well as from nonstimulated microglia. Further study using a specific conventional PKC (cPKC) activator, 1-oleoyl-2-acetyl-sn-glycerol (OAG), and a specific cPKC inhibitor, Gö 6976, suggested that PKC-delta and/or -epsilon is involved in uPA release. As opposed to the apoptotic pathway, however, no activation of c-Jun N-terminal kinase and nuclear factor kappa B was observed in C8-ceramide-stimulated microglia. The findings suggest that uPA release from microglia is regulated by a mechanism in which PKC-delta and/or -epsilon are activated and further signals are transduced subsequently.

Phylogenetic relation of lungfish indicated by the amino acid sequence of myelin DM20.

The cDNA of lungfish Protopterus annectens myelin DM20 was cloned, and the complete amino acid sequence of Protopterusannectens DM20 was deduced. When five possible phylogenetic trees were tested for the DM20 sequences, the maximum likelihood method supported tree 1 [((tetrapods, lungfish), coelacanth), zebrafish, shark] or tree 5 [(tetrapods, lungfish), (coelacanth, zebrafish), shark]. Both tree 1 and tree 5 indicate that lungfish is phylogenetically the closest to tetrapods among the living fishes.

Pyk2 and Syk participate in functional activation of granulocytic HL-60 cells in a different manner.

The roles of the protein tyrosine kinases Pyk2 (also called RAFTK or CAK beta) and Syk in the process of functional activation of human myeloid cells were examined. During granulocytic differentiation of HL-60 cells with dimethyl sulfoxide (DMSO), the amounts of Pyk2 and beta2 integrin increased, whereas the amount of Syk was abundant before differentiation and did not change during differentiation. When the granulocytic cells were stimulated with N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), tyrosine phosphorylation of Pyk2 occurred promptly and subsequent association of Pyk2 with beta2 integrin was detected. In contrast, Syk was not tyrosine phosphorylated by fMLP stimulation but constitutively associated with beta2 integrin. Stimulation with fMLP also caused the alteration of beta2 integrin to an activated form, a finding that was confirmed by the observation of fMLP-induced cell attachment on fibrinogen-coated dishes and inhibition of this attachment by pretreatment with anti-beta2 integrin antibody. Cell attachment to fibrinogen caused the enhanced tyrosine phosphorylation of Pyk2 and the initial tyrosine phosphorylation of Syk, which was also inhibited by pretreatment with anti-beta2 integrin antibody. In vitro kinase assays revealed that Pyk2 and Syk represented kinase activities to induce tyrosine phosphorylation of several molecules in the anti-beta2 integrin immunoprecipitates of the attached cells. These results showed that Pyk2 is involved in the functional activation of granulocytic cells in 2 signaling pathways: an fMLP receptor-mediated "inside-out" signaling pathway that might cause beta2 integrin activation and a subsequent beta2 integrin-mediated "outside-in" signaling pathway. Syk was activated in relation to cell attachment to fibrinogen as a result of "outside-in" signaling, although it was already associated with beta2 integrin before fMLP stimulation. (Blood. 2000;96:1733-1739)

Anti-HPA-5b-induced neonatal alloimmune thrombocytopenia: antibody titre as a predictor. Collaborative Study Group.

Anti-HPA-5b is the most commonly found platelet-specific antibody among pregnant women, but it does not cause severe fetal-neonatal alloimmune thrombocytopenia in the majority of affected infants. However, as the sequelae of the affected children may become severe, it is necessary to identify the risk factors for neonatal alloimmune thrombocytopenia. Of 21 354 consecutive pregnant women, 138 [0.65%; 95% confidence interval (CI) 0.54-0.75%], corresponding to 13.2% of the 1049 HPA-5b- women calculated by the gene frequency, were positive for anti-HPA-5b at the first trimester. Anti-HPA-5b was titrated in specimens obtained at the third trimester and antibody-positive women and their neonates were HPA-5 genotyped. Platelet counts in cord blood and 3 d after birth were assessed in the infants born to these mothers. Chi-square analysis showed no significant relationship between the titres of maternal antibody to HPA-5b and the number of pregnancies. There was a significant difference in platelet counts at d 3 between neonates who were compatible (267 x 109/l) or incompatible (220 x 109/l, P < 0.05) with maternal anti-HPA-5b. HPA-5b antibody titres >/= 64 were related to the development of thrombocytopenia (< 150 x 109/l) in neonates 1 d and 3 d after birth. A high titre (>/= 64) had a positive predictive value of 50% for thrombocytopenia 3 d after birth when the infant was HPA-5b+ and a negative predictive value of 100%. These results indicate that a high titre (>/= 64) of anti-HPA-5b is associated with a higher risk of neonatal thrombocytopenia, even if anti-HPA-5b-induced severe thrombocytopenia rarely develops.

[A case of Down's syndrome associated with testicular seminoma].

Down's syndrome is an inherited disorder caused by trisomy of chromosome 21. In patients with Down's syndrome, an increased risk of leukemia has been observed. Recently, the coincidence of testicular cancer with this syndrome has been also emphasized. We present a case of Down's syndrome associated with testicular seminoma. This is the 19th case of Down's syndrome associated with testicular tumor in Japan.

Helicobacter pylori CagA protein can be tyrosine phosphorylated in gastric epithelial cells.

Attachment of Helicobacter pylori to gastric epithelial cells induces various cellular responses, including the tyrosine phosphorylation of an unknown 145-kD protein and interleukin 8 production. Here we show that this 145-kD protein is the cagA product of H. pylori, an immunodominant, cytotoxin-associated antigen. Epithelial cells infected with various H. pylori clinical isolates resulted in generation of tyrosine-phosphorylated proteins ranging from 130 to 145 kD in size that were also induced in vitro by mixing host cell lysate with bacterial lysate. When epithelial cells were infected with [(35)S]methionine-labeled H. pylori, a radioactive 145-kD protein was detected in the immunoprecipitates with antiphosphotyrosine antibody or anti-CagA (cytotoxin-associated gene A) antibody. Consistently, the 145-kD protein recognized by the anti-CagA and antiphosphotyrosine antibodies was induced in epithelial cells after infection of wild-type H. pylori but not the cagA::Km mutant. Furthermore, the amino acid sequence of the phosphorylated 145-kD protein induced by H. pylori infection was identical to the H. pylori CagA sequence. These results reveal that the tyrosine-phosphorylated 145-kD protein is H. pylori CagA protein, which may be delivered from attached bacteria into the host cytoplasm. The identification of the tyrosine-phosphorylated protein will thus provide further insights into understanding the precise roles of CagA protein in H. pylori pathogenesis.

Brain net unidirectional uptake of alpha-[14c]methyl-L-tryptophan (alpha-MTrp) and its correlation with regional serotonin synthesis, tryptophan incorporation into proteins, and permeability surface area products of tryptophan and alpha-MTrp.

The uptake and trapping constants for labeled tryptophan (Trp) via the serotonin (5-hydroxytryptamine; 5-HT) metabolic pathway and for the incorporation of Trp into proteins, and alpha-[14C]methyl-L-tryptophan (alpha-MTrp) were measured. Measurements were done in rats treated with either saline or probenecid (200 mg/kg). In addition, the blood-brain barrier (BBB) permeability surface area products for Trp (PS(T)) and alpha-MTrp (PSalpha) were measured in normal rats. The results suggest that, in both groups of rats, there is a highly significant correlation (p < 0.05; Pearson Product Moment Correlation (PPMC) between the brain uptake and trapping constants for alpha-MTrp and those of Trp via the 5-HT metabolic pathway, but there is no significant correlation (p > 0.05; PPMC) between either of these constants and the PS products of either compound. There is also no significant correlation (p > 0.05; PPMC) between the constant for the Trp incorporation into proteins with any of the other parameters. For all parameters, except Trp incorporation into proteins (alpha-MTrp is not incorporated into proteins), there was a highly significant correlation (p < 0.001) between the quantities measured for Trp and alpha-MTrp. The data presented here strongly suggests that the brain uptake and trapping of alpha-MTrp relates to brain 5-HT synthesis, and does not relate to the BBB transport or protein incorporation of Trp. On the basis of these results, as well as those previously reported, we concluded that trapping (unidirectional uptake) of alpha-MTrp can be converted to the 5-HT synthesis rates in the brain. From this also follows that labeled alpha-MTrp is a good tracer for in vivo evaluation of the brain 5-HT synthesis.

Acute and chronic D-fenfluramine treatments have different effects on serotonin synthesis rates in the rat brain: an autoradiographic study.

The effects of acute and chronic treatments with D-fenfluramine on the regional rates of serotonin (5-hydroxy-tryptamine; 5-HT) synthesis were investigated using the alpha-[14C]methyl-L-tryptophan (alpha-[14C]MTrp) autoradiographic method. In the first series of experiments, acute D-fenfluramine treatment (5 mg/kg; i.p.) given 20 min before the tracer injection significantly (p<0.05) decreased 5-HT synthesis in the dorsal raphe, and significantly (p<0.05) increased the rates in the cerebral cortices and caudate nucleus, when compared to the rates in the control rats (saline treated). In a second series of experiments, following a 7-day treatment with D-fenfluramine (5 mg/kg/day; i.p.), a significant (p<0.05) decrease of 5-HT synthesis, in the dorsal raphe was observed, and significant (p<0.05) increases were observed in the hypothalamus, the dorsal thalamus, the medial and lateral geniculate body and some brain stem regions (locus ceruleus, inferior and superior colliculus). No significant changes were observed in the cerebral cortices.