Temperature sensitization of liposomes by use of N-isopropylacrylamide copolymers with varying transition endotherms

Three kinds of copolymers of N-isopropylacrylamide (NIPAM) with the same conformational transition temperature and varying transition endotherms were synthesized with N-acryloylpyrrolidine (APr), N,N-dimethylacrylamide (DMAM), and N-isopropylmethacrylamide (NIPMAM) as the comonomers. Two dodecyl groups were incorporated into the termini of these copolymers as an anchor for the fixation to a liposomal membrane. Egg yolk phosphatidylcholine liposomes having these copolymers were prepared and their temperature-sensitive contents release and association properties were investigated. While these copolymer exhibited a conformational transition at ca. 40 degrees C, DeltaH for the transition increased in the order of poly(APr-co-NIPAM) < poly(DMAM-co-NIPAM) < poly(NIPMAM-co-NIPAM). The liposomes containing poly(NIPMAM-co-NIPAM) showed a drastic release enhancement of entrapped calcein above the transition temperature, whereas the liposomes with poly(DMAM-co-NIPAM) and those with poly(APr-co-NIPAM) exhibited moderate and slight enhancement of calcein release above that temperature, respectively. On the contrary, the liposomes containing poly(APr-co-NIPAM) showed significant aggregation above the transition temperature, but the aggregation was hardly observed for the liposomes having poly(NIPMAM-co-NIPAM), indicating that poly(APr-co-NIPAM) more efficiently made the liposome surface hydrophobic. Thus, we concluded that the copolymer with a large DeltaH is suitable for obtaining functional liposomes with a temperature-sensitive contents release property, whereas the copolymer with a small DeltaH is appropriate for preparing functional liposomes with a temperature-sensitive surface property.     

Cyclic ADP-ribose, a putative Ca2+-mobilizing second messenger, operates in submucosal gland acinar cells

Cyclic ADP-ribose (cADPR), a putative Ca(2+)-mobilizing second messenger, has been reported to operate in several mammalian cells. To investigate whether cADPR is involved in electrolyte secretion from airway glands, we used a patch-clamp technique, the measurement of microsomal Ca(2+) release, quantification of cellular cADPR, and RT-PCR for CD38 mRNA in human and feline tracheal glands. cADPR (>6 microM), infused into the cell via the patch pipette, caused ionic currents dependent on cellular Ca(2+). Infusions of lower concentrations (2-4 microM) of cADPR or inositol 1,4,5-trisphosphate (IP(3)) alone were without effect on the baseline current, but a combined application of cADPR and IP(3) mimicked the cellular response to low concentrations of acetylcholine (ACh). Microsomes derived from the isolated glands released Ca(2+) in response to both IP(3) and cADPR. cADPR released Ca(2+) from microsomes desensitized to IP(3) or those treated with heparin. The mRNA for CD38, an enzyme protein involved in cADPR metabolism, was detected in human tissues, including tracheal glands, and the cellular content of cADPR was increased with physiologically relevant concentrations of ACh. We conclude that cADPR, in concert with IP(3), operates in airway gland acinar cells to mobilize Ca(2+), resulting in Cl(-) secretion.     

Immunoglobulin treatment suppresses atherosclerosis in apolipoprotein E-deficient mice via the Fc portion

Atherosclerosis is associated with immune activation. Immunoglobulin is used for the treatment of immune-mediated diseases. The mechanisms and importance of the Fc portion of immunoglobulin upon experimental atherosclerosis in apolipoprotein E-deficient mice were examined. Experimental atherosclerosis was induced in mice fed a high-fat diet containing 0.3% cholesterol. Over 8, 12, and 16 wk, on alternate days, mice were treated with an intraperitoneal injection of either 1 g.kg-1.day-1 of human intact immunoglobulin or F(ab')2 fragments of human immunoglobulin. Fatty streak formation and fibrofatty plaques were markedly suppressed in mice that received intact immunoglobulin for 8, 12, and 16 wk. In contrast, atherosclerotic lesions were not ameliorated in mice that received F(ab')2 fragments. Immunohistochemical analysis revealed that macrophage accumulation in the fatty streak lesions was suppressed in mice received intact immunoglobulin but not in those that received F(ab')2 fragments. In addition, the cytotoxic activities of splenocytes from immunoglobulin-treated mice, but not from F(ab')2 fragment-treated mice, were significantly suppressed compared with those from human serum albumin-treated mice. Differences in lesion area did not correlate with any significant alterations in serum lipid levels. Immunoglobulin therapy markedly suppressed atherosclerosis due to Fc receptor-mediated anti-inflammatory and immunomodulating actions. The antiatherosclerotic effects of immunoglobulin may be related to the suppression of cytotoxic activity of atherogenic T cells and the reduction of macrophage accumulation in the lesions.     

Patch establishment and development of a clonal plant,Polygonum cuspidatum,on Mount Fuji

Microsatellite analysis was used to investigate the patch establishment and development of Polygonum cuspidatum Sieb. et Zucc, a clonal herbaceous plant that dominates the primary succession on the southeast slope of Mount Fuji. Genotypes of P. cuspidatum in 155 patches at the study site differed from each other. This indicates that P. cuspidatum patches are initially established by seed dispersed on the bare scoria field, and not by clonal rhizome extension. Genetic differentiation was estimated using the FST values between subpopulations at the study site. There was almost no genetic differentiation between subpopulations, indicating the presence of massive gene flow. The pollen fathers of seeds and maternal genets of current-year seedlings were inferred from the microsatellite allele composition by a simple exclusion method. The wide, random distribution of pollen fathers suggests that pollen dispersal occurs over a broad area. Maternal analysis showed a tendency for seed dispersal to be biased to the area nearby and down slope from the mother plants. Patch establishment under massive gene flow may result from such pollen and seed dispersal. To understand the process of patch development, aerial photographs taken from 1962 to 1999 were compared, and then genets in each of 36 patches were identified from the microsatellite genotypes of P. cuspidatum shoots. The comparison of aerial photographs showed that most of the patches enlarged each year and that some neighbouring patches combined during growth. Genet analysis demonstrated a high correlation between patch area and the area of the largest genet within it, and that new genets were recruited at the patch periphery. These findings indicate that both vegetative and sexual reproduction, i.e. rhizome extension and the establishment of new seedlings, contribute to the development of P. cuspidatum patches.     

Genetic structure and reproduction dynamics of Salix reinii during primary succession on Mount Fuji,as revealed by nuclear and chloroplast microsatellite analysis

The early stage of volcanic desert succession is underway on the southeastern slope of Mount Fuji. We used markers of nuclear microsatellites (simple sequence repeats; SSR) and chloroplast microsatellites (cpSSR) to investigate the population genetic structure and reproduction dynamics of Salix reinii, one of the dominant pioneer shrubs in this area. The number of S. reinii genets in a patch and the area of the largest genet within the patch increased with patch area, suggesting that both clonal growth and seedling recruitment are involved in the reproduction dynamics of S. reinii. Five polymorphic cpSSR markers were developed for S. reinii by sequencing the noncoding regions between universal sequences in the chloroplast genome. Nineteen different cpSSR haplotypes were identified, indicating that S. reinii pioneer genets were created by the long-distance dispersal of seeds originating from different mother genets around the study site, where all vegetation was destroyed during the last eruption. Furthermore, the clustered distributions of different haplotypes within each patch or plot suggested that newly colonized genets tended to be generated from seeds dispersed near the initially established mother genets. These results revealed that the establishment of the S. reinii population on the southeastern slope of Mount Fuji involved two sequential modes of seed dispersal: long-distance dispersal followed by short-distance dispersal.     

Involvement of c-Jun N-terminal kinase in amyloid precursor protein-mediated neuronal cell death

Amyloid precursor protein (APP), the precursor of Abeta, has been shown to function as a cell surface receptor that mediates neuronal cell death by anti-APP antibody. The c-Jun N-terminal kinase (JNK) can mediate various neurotoxic signals, including Abeta neurotoxicity. However, the relationship of APP-mediated neurotoxicity to JNK is not clear, partly because APP cytotoxicity is Abeta independent. Here we examined whether JNK is involved in APP-mediated neuronal cell death and found that: (i) neuronal cell death by antibody-bound APP was inhibited by dominant-negative JNK, JIP-1b and SP600125, the specific inhibitor of JNK, but not by SB203580 or PD98059; (ii) constitutively active (ca) JNK caused neuronal cell death and (iii) the pharmacological profile of caJNK-mediated cell death closely coincided with that of APP-mediated cell death. Pertussis toxin (PTX) suppressed APP-mediated cell death but not caJNK-induced cell death, which was suppressed by Humanin, a newly identified neuroprotective factor which inhibits APP-mediated cytotoxicity. In the presence of PTX, the PTX-resistant mutant of Galphao, but not that of Galphai, recovered the cytotoxic action of APP. These findings demonstrate that JNK is involved in APP-mediated neuronal cell death as a downstream signal transducer of Go.     

Nerve growth factor-induced glutamate release is via p75 receptor,ceramide, and Ca(2+) from ryanodine receptor in developing cerebellar neurons

Very little is known about the contribution of a low affinity neurotrophin receptor, p75, to neurotransmitter release. Here we show that nerve growth factor (NGF) induced a rapid release of glutamate and an increase of Ca2+ in cerebellar neurons through a p75-dependent pathway. The NGF-induced release occurred even in the presence of the Trk inhibitor K252a. The release caused by NGF but not brain-derived neurotrophic factor was enhanced in neurons overexpressing p75. Further, after transfection of p75-small interfering RNA, which down-regulated the endogenous p75 expression, the NGF-induced release was inhibited, suggesting that the NGF-induced glutamate release was through p75. We found that the NGF-increased Ca2+ was derived from the ryanodine-sensitive Ca2+ receptor and that the NGF-increased Ca2+ was essential for the NGF-induced glutamate release. Furthermore, scyphostatin, a sphingomyelinase inhibitor, blocked the NGF-dependent Ca2+ increase and glutamate release, suggesting that a ceramide produced by sphingomyelinase was required for the NGF-stimulated Ca2+ increase and glutamate release. This action of NGF only occurred in developing neurons whereas the brain-derived neurotrophic factor-mediated Ca2+ increase and glutamate release was observed at the mature neuronal stage. Thus, we demonstrate that NGF-mediated neurotransmitter release via the p75-dependent pathway has an important role in developing neurons.     

A novel DNA polymerase inhibitor and a potent apoptosis inducer: 2-mono-O-acyl-3-O-(alpha-D-sulfoquinovosyl)-glyceride with stearic acid

Sulfo-glycolipids in the class of sulfoquinovosyl diacylglycerol (SQDG) including the stereoisomers are potent inhibitors of DNA polymerase alpha and beta. However, since the alpha-configuration of SQDG with two stearic acids (alpha-SQDG-C(18)) can hardly penetrate cells, it has no cytotoxic effect. We tried and succeeded in making a permeable form, sulfoquinovosyl monoacylglycerol with a stearic acid (alpha-SQMG-C(18)) from alpha-SQDG-C(18) by hydrolysis with a pancreatic lipase. alpha-SQMG-C(18) inhibited DNA polymerase activity and was found to be a potent inhibitor of the growth of NUGC-3 cancer cells. alpha-SQMG-C(18) arrested the cell cycle at the G1 phase, and subsequently induced severe apoptosis. The arrest was correlated with an increased expression of p53 and cyclin E, indicating that alpha-SQMG-C(18) induced cell death through a p53-dependent apoptotic pathway.     

Pancreatoblastoma. A case report with special emphasis on squamoid corpuscles with optically clear nuclei rich in biotin

BACKGROUND: Pancreatoblastoma (PBL) is a rare neoplasm that generally occurs in the pediatric age group and shows unique histopathology, including squamoid corpuscles that may contain tumor cells with optically clear nuclei (OCN) rich in biotin. In the English-language literature there have been two reports on the cytology of PBL, but neither of them refers to the cytologic features of squamoid corpuscles. CASE: A 3-year-old boy with nausea and general fatigue was referred to our center. Imaging studies showed an approximately 7.5-cm, left-sided abdominal mass and multiple metastases in the lung. The abdominal mass was biopsied, and its histology showed solid cellular nests with occasional acinar differentiation and squamoid corpuscles. Imprint cytology of the biopsied sample displayed cellular epithelial nests with focal acinar structures and foci composed of larger cells with a low nuclear/cytoplasmic ratio. These foci contained a few tumor cells with biotin-rich OCN and were determined to be squamoid corpuscles. CONCLUSION: Detection of occasional squamoid corpuscles with biotin-rich OCN can be useful in making a diagnosis of PBL on cytologic samples.     

Large-scale search of SNPs for type 2 DM susceptibility genes in a Japanese population

The etiology of type 2 diabetes (DM) is polygenic. We investigated here genes and polymorphisms that associate with DM in the Japanese population. Single-nucleotide polymorphisms (SNPs) of 398 derived from 120 candidate genes were examined for association with DM in a population-based case-control study. The study group consisted of 148 cases and 227 controls recruited from Funagata, Japan. No evident subpopulation structure was detected for the tested population. The association tests were conducted with standard allele positivity tables (chi(2) tests) between SNP genotype frequency and case-control status. The independent association of the SNPs from serum triglyceride levels and body mass index was examined by multiple logistic regression analysis. A value of P<0.01 was accepted as statistically significant. Six genes (met proto-oncogene, ATP-binding cassette transporter A1, fatty acid binding protein 2, LDL receptor defect C complementing, aldolase B, and sulfonylurea receptor) were shown to be associated with DM.