Terminal N-Acetylgalactosamine-Specific Leguminous Lectin from Wisteria japonica as a Probe for Human Lung Squamous Cell Carcinoma

Millettia japonica was recently reclassified into the genus Wisteria japonica based on chloroplast and nuclear DNA sequences. Because the seed of Wisteria floribunda expresses leguminous lectins with unique N-acetylgalactosamine-binding specificity, we purified lectin from Wisteria japonica seeds using ion exchange and gel filtration chromatography. Glycan microarray analysis demonstrated that unlike Wisteria floribunda and Wisteria brachybotrys lectins, which bind to both terminal N-acetylgalactosamine and galactose residues, Wisteria japonica lectin (WJA) specifically bound to both α- and β-linked terminal N-acetylgalactosamine, but not galactose residues on oligosaccharides and glycoproteins. Further, frontal affinity chromatography using more than 100 2-aminopyridine-labeled and p-nitrophenyl-derivatized oligosaccharides demonstrated that the ligands with the highest affinity for Wisteria japonica lectin were GalNAcβ1-3GlcNAc and GalNAcβ1-4GlcNAc, with K _a values of 9.5 × 10⁴ and 1.4 × 10⁵ M^-1, respectively. In addition, when binding was assessed in a variety of cell lines, Wisteria japonica lectin bound specifically to EBC-1 and HEK293 cells while other Wisteria lectins bound equally to all of the cell lines tested. Wisteria japonica lectin binding to EBC-1 and HEK293 cells was dramatically decreased in the presence of N-acetylgalactosamine, but not galactose, mannose, or N-acetylglucosamine, and was completely abrogated by β-hexosaminidase-digestion of these cells. These results clearly demonstrate that Wisteria japonica lectin binds to terminal N-acetylgalactosamine but not galactose. In addition, histochemical analysis of human squamous cell carcinoma tissue sections demonstrated that Wisteria japonica lectin specifically bound to differentiated cancer tissues but not normal tissue. This novel binding characteristic of Wisteria japonica lectin has the potential to become a powerful tool for clinical applications.     

Apoptotic cells induce tolerance by generating helpless CD8+ T cells that produce TRAIL

The decision to generate a productive immune response or immune tolerance following pathogenic insult often depends on the context in which T cells first encounter Ag. The presence of apoptotic cells favors the induction of tolerance, whereas immune responses generated with necrotic cells promote immunity. We have examined the tolerance induced by injection of apoptotic cells, a system in which cross-presentation of Ag associated with the dead cells induces CD8+ regulatory (or suppressor) T cells. We observed that haptenated apoptotic cells induced CD8+ suppressor T cells without priming CD4+ T cells for immunity. These CD8+ T cells transferred unresponsiveness to naive recipients. In contrast, haptenated necrotic cells stimulated immunity, but induced CD8+ suppressor T cells when CD4+ T cells were absent. We further found that CD8+ T cells induced by these treatments displayed a "helpless CTL" phenotype and suppress the immune response by producing TRAIL. Animals deficient in TRAIL were resistant to tolerance induction by apoptotic cells. Thus, the outcome of an immune response taking place in the presence of cell death can be determined by the presence of CD4+-mediated Th cell function.     

Floral development of an asexual and female-like mutant carrying two deletions in gynoecium-suppressing and stamen-promoting functional regions on the Y chromosome of the dioecious plant Silene latifolia

Sexual dimorphism is controlled by genes on the Y chromosome in the dioecious plant Silene latifolia. K034 is the first mutant with female flowers and asexual flowers in one individual. Its stamens are suppressed completely, and its gynoecium exhibits two suppression patterns. One gynoecium resembles a thin rod, as in wild-type males (asexual flower); the other is imperfectly suppressed, having 1-3 carpels (female-like flower). The ratio of these patterns was 9 : 1. To exclude the possibility of chimerism in K034, we crossed a female-like flower of K034 with a wild-type male. Progeny obtained from this crossing had asexual and female-like flowers in one individual. This two-flower-type phenotype was inherited without separating. To examine the identity of flower organs in K034, we analyzed the development of asexual and female-like flowers using scanning electron microscopy and in situ hybridization with SLM1 and SLM2 (orthologs of AGAMOUS and PISTILLATA, respectively) as probes. Mitotic spreads of root tip chromosomes from hairy root cultures showed that K034 had 25 chromosomes. Fluorescent in situ hybridization analysis, using a subtelomeric repetitive sequence (KpnI subfamily) as a probe, indicated that K034 possessed two X chromosomes and one Y chromosome (Y(d)), of which Y(d) had been rearranged to lose the pseudoautosomal region (PAR). PCR analysis using Y-specific sequence-tagged site (STS) markers clarified that Y(d) of K034 had two other deletions in gynoecium-suppressing and stamen-promoting regions. It is reasonable to suggest that these sex chromosomal abnormalities resulted in two abnormal sexual phenotypes: the asexual and imperfect female (female-like) flowers in K034.     

Human Apolipoprotein E Resequencing by Proteomic Analysis and Its Application to Serotyping

Background

Apolipoprotein E (ApoE) typing is considered important because of the association between ApoE and Alzheimer’s disease and familial dyslipidemia and is currently performed by genetic testing (APOE genotyping). ApoE levels in plasma and serum are clinically determined by immunoassay.

Methods

Combining an ApoE immunoassay reagent with proteomic analysis using an Orbitrap mass spectrometer, we attempted to resequence ApoE from trace amounts of serum for typing (serotyping). Most (24 of 33) ApoE mutant proteins registered to date with Online Mendelian Inheritance in Man, such as ApoE2 and ApoE4, involve lysine and arginine mutations. Digestion of mutant ApoE with trypsin will thus result in fragments that differ substantially from wild-type ApoE3 in terms of mass, making serotyping ideally suited to mass spectrometry analysis.

Results

The mean coverage of the amino acid sequence of full-length ApoE was 91.6% in the protein resequence. Residues 112 and 158 (which are mutated in ApoE2 and ApoE4) were covered in all samples, and the protein sequences were used for serotyping. Serotypes including all heterozygous combinations (ApoE2/E3, E2/E4, E3/E4) corresponded exactly to the APOE genotyping results in each of the subjects.

Conclusion

Our novel ApoE serotyping method with protein resequencing requires no synthesis of stable isotope-labeled peptides or genome analysis. The method can use residual blood from samples collected for routine clinical tests, thus enabling retrospective studies with preserved body fluids. The test could be applied to samples from subjects whose DNA is unavailable. In future studies, we hope to demonstrate the capability of our method to detect rare ApoE mutations.     

A novel adipocytokine,omentin, inhibits monocrotaline-induced pulmonary arterial hypertension in rats

Omentin is a novel adipocytokine mainly expressed in visceral rather than subcutaneous adipose tissue. Several epidemiological studies demonstrated the negative relationship between blood omentin level and occurrence of obesity, type 2 diabetes and hypertension. Increases of inflammatory responses, contractile reactivity and structural remodeling of vascular wall contribute to hypertension development. Our in vitro studies previously demonstrated that omentin inhibited those hypertension-related pathological processes. In addition, our in vivo study demonstrated that intravenously injected omentin acutely inhibited agonists-induced increases of blood pressure in rats. However, the chronic effects of omentin on hypertension development are not determined. In the present study, we tested the hypothesis that chronic omentin treatment may inhibit pulmonary arterial (PA) hypertension (PAH). PAH was induced by a single intraperitoneal injection of monocrotaline (MCT: 60 mg/kg) to rats. Omentin (18 μg/kg/day) was intraperitoneally treated for 14 days. Chronic omentin treatment inhibited MCT-induced increases in PA pressure. Omentin inhibited MCT-induced right ventricular hypertrophy as well as increase of lung to body weight ratio. Histologically, omentin inhibited MCT-induced PA hyperplasia. Further, omentin inhibited the impairment of both endothelium-dependent and -independent relaxations mediated by acetylcholine and sodium nitroprusside, respectively. In conclusion, we for the first time demonstrate that chronic omentin treatment inhibits MCT-induced PAH in rats via inhibiting vascular structural remodeling and abnormal contractile reactivity.     

Blockade of the ERK pathway markedly sensitizes tumor cells to HDAC inhibitor-induced cell death

Constitutive activation of the extracellular signal-regulated kinase (ERK) pathway is associated with the neoplastic phenotype of a large number of human tumor cells. Although specific blockade of the ERK pathway by treating such tumor cells with potent mitogen-activated protein kinase/ERK kinase (MEK) inhibitors completely suppresses their proliferation, it by itself shows only a modest effect on the induction of apoptotic cell death. However, these MEK inhibitors markedly enhance the efficacy of histone deacetylase (HDAC) inhibitors to induce apoptotic cell death: such an enhanced cell death is observed only in tumor cells in which the ERK pathway is constitutively activated. Co-administration of MEK inhibitor markedly sensitizes tumor cells to HDAC inhibitor-induced generation of reactive oxygen species, which appears to mediate the enhanced cell death induced by the combination of these agents. These results suggest that the combination of MEK inhibitors and HDAC inhibitors provides an efficient chemotherapeutic strategy for the treatment of tumor cells in which the ERK pathway is constitutively activated.     

Progress of eutrophication and change of chironomid fauna in Lake Yamanakako,Japan

Benthic macroinvertebrate distribution was studied on April 9, 1994, and March 2, 2003, in Lake Yamanakako. The average density of the benthic community for the entire lake was 3168ind.m^–2, comprising principally oligochaetes (41.0%) and chironomids (59.0%) in 1994. In 2003, the benthic community for the entire lake was 1847ind.m^–2, principally consisting of oligochaetes (69.9%) and chironomids (30.1%). In 1994, the larval density of Propsilocerus akamusi was 3.5 times that of Chironomus nipponensis and in 2003 the figure was 5.7 times. However, the larval biomass of P. akamusi was 2.1 times greater than that of C. nipponensis in 1994 and 2.8 times greater in 2003. The larval density of Tanypodinae decreased drastically, by about 12-fold, from 1994 to 2003. P. akamusi larvae were particularly abundant at the lake center in 1994, but they inhabited the entire lake bottom in 2003. P. akamusi density was closely related to water depth and ignition loss. C. nipponensis larvae also showed the widest distribution pattern in 2003, whereas their larvae had inhabited the northeastern parts and the lake center in 1994. Recently, the number of C. nipponensis larvae in Lake Yamanakako is tending to decrease, whereas that of P. akamusi larvae is increasing, suggesting ongoing eutrophication.     

Ultrastructure of Thraustochytrium sp. zoospores

Summary Acid phosphatase distribution in the biflagellate zoospores of a marine fungus Thraustochytrium, resembling T. motivum Goldstein, was examined utilizing ultrastructural cytochemistry. Acid phosphatase activity was found in the Golgi saccules, Golgi vesicles, multivesicular bodies, endoplasmic reticulum, and autophagic vacuoles.Extensive autolysis of cellular structures occurs in the zoospores. Organelles or portions of the cytoplasm are segregated from the rest of the cytoplasm by acid phosphatase-positive vesicles and lamellae. These vesicles and lamellae coalesce around a portion of cytoplasm forming an enclosing double membraned sac. One of the membranes, probably the inner, is disrupted, releasing the hydrolytic enzymes which initiate digestion of the enclosed cytoplasm. These cytolysomes eventually fuse with larger cytolysomes where digestion is presumably completed. The final fate of the digestive residues and the large cytolysomes has not been determined.Contribution No. 501, Virginia Institute of Marine Science, Gloucester Point, Virginia 23062, U.S.A.Supported in part by the Oceanographic Section, National Science Foundation, Grant # GA-31014, to Dr. Frank O. Perkins.     

Auxin-gibberellin relationships in their effects on hypocotyl elongation of light-grown cucumber seedlings. Responses of sections to auxin, gibberellin and sucrose

The sensitivity of light-grown cucumber hypocotyl sections toIAA and GA₃ depends on the degree of aging of the tissue. Agreater response to GA₃ was obtained with young tissue, whilethat to IAA was obtained with relatively old tissue. The responseto IAA reached a maximum at about 15 hr of incubation; the youngerthe tissue the earlier the time of maximum response. The responseto GA₃ continued for more than 70 hr with a constant growthrate. Very young tissue started to respond to GA₃ without lagtime; the older the tissue the later the start of the response. Sucrose (2%) inhibited IAA-induced elongation, while there wasa distinct synergism between GA₃ and sucrose. The promotiveeffect of sucrose on GA₃-induced elongation was also obtainedwhen sections were pretreated with sucrose, then transferredto GA₃. Mannitol (1%) strongly inhibited IAA-induced elongation,but not GA₃-induced elongation. (Received December 6, 1972; )     

Effects of light on auxin-induced elongation of light-grown cucumber hypocotyl sections

The effect of light on IAA-induced elongation of light-growncucumber hypocotyl sections was examined. There was no differencein the optimal concentration of IAA between light and darknessand the biphasic pattern of IAA-induced elongation also wasobtained in both states. Elongation of the first phase was notaffected by light, but that of the second phase was influencedby the light condition of the first phase; dark incubation forthe first 3 hr resulted in a larger IAA-induced elongation inthe second phase. For the maximum IAA-induced elongation, atleast 3 hr of light exposure in the second phase was necessary.Simultaneously applied photosynthetic inhibitors, DCMU, CMUand o-phenanthroline, synergistically enhanced IAA-induced elongationin light, but not in the dark. They were not effective whenIAA treatment preceded the treatment with them. (Received April 3, 1978; )