Gate-Tunable Atomically Thin Lateral MoS2 Schottky Junction Patterned by Electron Beam.

Among atomically thin two-dimensional (2D) materials, molybdenum disulfide (MoS2) is attracting considerable attention because of its direct bandgap in the 2H-semiconducting phase. On the other hand, a 1T-metallic phase has been revealed, bringing complementary application. Recently, thanks to top-down fabrication using electron beam (EB) irradiation techniques, in-plane 1T-metal/2H-semiconductor lateral (Schottky) MoS2 junctions were demonstrated, opening a path toward the co-integration of active and passive two-dimensional devices. Here, we report the first transport measurements evidencing the formation of a MoS2 Schottky barrier (SB) junction with barrier height of 0.13-0.18 eV created at the interface between EB-irradiated (1T)/nonirradiated (2H) regions. Our experimental findings, supported by state-of-the-art simulation, reveal unique device fingerprint of SB-based field-effect transistors made from atom-thin 1T layers.

Intracytoplasmic sperm injection. Accomplishments and qualms.

AIM: Intracytoplasmic sperm injection (ICSI) is now the preferred technique for treatment of male factor infertility and many children have been born worldwide. However, concerns about the risk of transmitting genetic defects and development of ICSI children have been raised. We report clinical outcome of ICSI in Cornell University and results of screening for genetic defects in ICSI parents and their children. METHODS: Pregnancy and obstetrical outcomes as well as congenital malformations were analyzed. Chromosomal karyotyping and Yq deletion assessments were performed on blood samples. In addition, medical and developmental outcome were assessed in 3 and 5 year old ICSI children. RESULTS: We have performed 8 575 ICSI cycles with ejaculated spermatozoa that resulted in a 75.4% fertilization and a 42.8% clinical pregnancy rates while for surgically retrieved specimen resulted in 66%, 48.8% respectively. The incidence of Y deletion was within the expected range for an infertile population, with identical deletions transmitted to the male offspring. There were no differences in cognitive, motor and behavioral development observed between ICSI children and these conceived naturally. CONCLUSION: The large majority of infertile men were treatable by ICSI, which resulted in the transmission of a specific abnormality but did not enhance the incidence of de novo deletions. There is no indication that ICSI children develop more congenital defects or express a lower psychomotor development that children conceived naturally. Nonetheless, genetic screening and counseling of couples undergoing ICSI would seem to be appropriate.

Application of intracytoplasmic sperm injection in assisted reproductive technologies.

Intracytoplasmic sperm injection (ICSI) entails the mechanical insertion of a chosen spermatozoon directly into the cytoplasm of an oocyte. Due to the consistent fertilization and pregnancy outcome, ICSI is routinely used to treat azoospermic patients where spermatozoa are retrieved by epididymal aspiration or testicular biopsy. Since male subfertility has been associated with a higher incidence of genomic defects, ranging from numerical chromosomal abnormalities to Yq microdeletions, concerns have been raised as to the risk of transmitting genetic defects to the offspring. Screening for such defects can provide invaluable information for appropriate counselling prior to ICSI treatment. In order to address these concerns, a follow-up of the children born after ICSI treatment was conducted.

Costimulatory signals distinctively affect CD20- and B-cell-antigen-receptor-mediated apoptosis in Burkitt's lymphoma/leukemia cells.

CD20 is a B-cell differentiation antigen and known to induce apoptosis in Burkitt's lymphoma/leukemia (BL) cells upon antibody-mediated crosslinking. We examined the biological effect of CD20 crosslinking on BL cell lines and observed that apoptosis induction is accompanied by activation of multiple caspases, including caspase-8, -9, -3, -2, and -7. Further investigation revealed a clear synergism between apoptosis mediated by CD20 and by B-cell antigen receptor (BCR). Examination of the effect of simultaneous crosslinking of other cell surface molecules with crosslinking of CD20 or BCR on apoptosis induction showed that these molecules had either a synergistic or inhibitory effect on induction of apoptosis. It is worth noting that some molecules had a different effect on CD20- and BCR-mediated apoptosis. Simultaneous crosslinking of the molecules CD10, CD22, CD72, and CD80 inhibited BCR-mediated apoptosis, but enhanced CD20-mediated apoptosis. Further studies revealed that regulation of CD20-induced apoptosis by other costimulatory molecules is achieved by modification of caspase activation. CD20-mediated apoptosis in BL cells may provide not only a model for understanding the mechanism regulating clonal selection of B cells but a new therapeutic strategy for BL patients.

Heparin-stimulated expression of extracellular-superoxide dismutase in human fibroblasts.

Extracellular-superoxide dismutase (EC-SOD) is the major SOD isozyme in the arterial wall and may be important for antioxidation capability of the vascular wall and normal vascular function. EC-SOD is expressed in various cell types in the vascular wall such as fibroblasts, smooth muscle cells and macrophages, and the synthesis of EC-SOD by human fibroblasts is known to be highly responsive to various inflammatory cytokines, although there is no response to oxidative stress. Heparin is a highly sulfated glycosaminoglycan with many functions such as antithrombotic, antilipemic and antiatherosclerotic effects. Another less well-known function of heparin is regulation of protein synthesis. In this study, we measured the induction of EC-SOD after treatment with heparin to understand the role of heparin in the antiatherosclerotic response of fibroblasts. Heparin induced EC-SOD expression at both the mRNA and protein levels. Heparin showed the greatest stimulatory effect and heparan sulfate showed moderate effects. The effect of chondroitin sulfate A was not clear. In contrast, desulfated heparin and chondroitin sulfate C did not increase EC-SOD expression. The stimulatory effect seemed to increase roughly with the degree of glycosaminoglycan sulfation. The enhanced expression of EC-SOD by heparin must contribute to the antiatherosclerotic effect of heparin.

Kinetic analysis of binding between Shiga toxin and receptor glycolipid Gb3Cer by surface plasmon resonance.

Shiga toxin (Stx) binds to the receptor glycolipid Gb3Cer on the cell surface and is responsible for hemolytic uremic syndrome. Stx has two isoforms, Stx1 and Stx2, and in clinical settings Stx2 is known to cause more severe symptoms, although the differences between the mechanisms of action of Stx1 and Stx2 are as yet unknown. In this study, the binding modes of these two isoforms to the receptor were investigated with a surface plasmon resonance analyzer to compare differences by real time receptor binding analysis. A sensor chip having a lipophilically modified dextran matrix or quasicrystalline hydrophobic layer was used to immobilize an amphipathic lipid layer that mimics the plasma membrane surface. Dose responsiveness was observed with both isoforms when either the toxin concentration or the Gb3Cer concentration was increased. In addition, this assay was shown to be specific, because neither Stx1 nor Stx2 bound to GM3, but both bound weakly to Gb4Cer. It was also shown that a number of fitting models can be used to analyze the sensorgrams obtained with different concentrations of the toxins, and the "bivalent analyte" model was found to best fit the interaction between Stxs and Gb3Cer. This shows that the interaction between Stxs and Gb3Cer in the lipid bilayer has a multivalent effect. The presence of cholesterol in the lipid bilayer significantly enhanced the binding of Stxs to Gb3Cer, although kinetics were unaffected. The association and dissociation rate constants of Stx1 were larger than those of Stx2: Stx2 binds to the receptor more slowly than Stx1 but, once bound, is difficult to dissociate. The data described herein clearly demonstrate differences between the binding properties of Stx1 and Stx2 and may facilitate understanding of the differences in clinical manifestations caused by these toxins.

Effect of interleukin-1 receptor antagonist on concanavalin A response of peripheral blood mononuclear cells from newborn calves.

The coexistence of interleukin (IL)-1beta with IL-1 receptor antagonist (ra) in bovine colostrum and the possibility of simultaneous transfer of these cytokines to neonates via colostrum have been demonstrated. In the present study, we investigated the effect of IL-1ra on the mitogenic response of calf peripheral blood mononuclear cells (PBMC) stimulated by concanavalin A (ConA), which was mediated by IL-1. Pretreatment of PBMC with recombinant bovine (rb) IL-1ra alone significantly suppressed the proliferation of ConA-stimulated cells. However, in the presence of rbIL-1beta, the suppressive activity of rbIL-1ra was counteracted. These results suggest that coexistence of IL-1ra with IL-1 in colostrum may have no effect on the activation of the neonatal immune system by IL-1beta.

Single-step method for purification of Shiga toxin-1 B subunit using receptor-mediated affinity chromatography by globotriaosylceramide-conjugated octyl sepharose CL-4B.

A new single-step purification method for Shiga toxin (Stx) was developed using receptor-mediated affinity chromatography, in which Gb3Cer (globotriaosylceramide) was conjugated to octyl Sepharose CL-4B as a carrier. This method achieves high yield and high purity in a small column on which Gb3Cer has been immobilized at high density. Using this affinity column, the Stx1 B subunit was purified with homogeneity by a one-step procedure from a crude extract of recombinant Stx1 B subunit-producing Escherichia coli. The purified Stx1 B subunit conserved a natural pentamer structure confirmed by gel filtration and sedimentation equilibrium analysis. Furthermore, the purified Stx1 B subunit was able to bind specifically to Gb3Cer expressed on Burkitt's lymphoma cells. This versatile purification method can be used to isolate various types of natural as well as recombinant Stx, facilitating fundamental studies of human diseases caused by this toxin.

High-performance liquid chromatographic assay of zonisamide in human plasma using a non-porous silica column.

A new method for measuring zonisamide (ZNS) in plasma by high-performance liquid chromatography was developed by using a 2-microm reversed-phase non-porous silica column. ZNS in plasma was first purified with a column extraction technique and injected onto the non-porous silica column. Calibration curve was linear over the concentration range of 1-80 microg/ml in plasma. The recoveries of ZNS added to plasma were more than 95.4% with the coefficient of variation less than 9.0%. We developed a rapid routine method using the non-porous silica column that was accurate and improved solvent consumption in the measurement of ZNS.

CD24 induces apoptosis in human B cells via the glycolipid-enriched membrane domains/rafts-mediated signaling system.

The glycosylphosphatidylinositol-anchored CD24 protein is a B cell differentiation Ag that is expressed on mature resting B cells but disappears upon Ag stimulation. We used Burkitt's lymphoma (BL) cells, which are thought to be related to germinal center B cells, to examine the biological effect of Ab-mediated CD24 cross-linking on human B cells and observed 1) induction of apoptosis in BL cells mediated by cross-linking of CD24; and 2) synergism between the cross-linking of CD24 and that of the B cell receptor for Ag in the effect on apoptosis induction. We also observed activation of mitogen-activated protein kinases following CD24 cross-linking, suggesting that CD24 mediates the intracellular signaling that leads to apoptosis in BL cells. Although CD24 has no cytoplasmic portion to transduce signals intracellularly, analysis of biochemically separated glycolipid-enriched membrane (GEM) fractions indicated enhanced association of CD24 and Lyn protein tyrosine kinase in GEM as well as increased Lyn kinase activity after CD24 cross-linking, suggesting that CD24 mediates intracellular signaling via a GEM-dependent mechanism. Specific microscopic cocapping of CD24 and Lyn, but not of other kinases, following CD24 cross-linking supported this idea. We further observed that apoptosis induction by cross-linking is a common feature shared by GEM-associated molecules expressed on BL cells, including GPI-anchored proteins and glycosphingolipids. CD24-mediated apoptosis in BL cells may provide a model for the cell death mechanism initiated by GEM-associated molecules, which is closely related to B cell receptor for Ag-mediated apoptosis.

Demonstration of a rhodopsin-retinochrome system in the stalk eye of a marine gastropod, Onchidium, by immunohistochemistry.

The stalk eye of Onchidium sp. (Gastropoda, Mollusca) is the principal photoreceptor in a multiple photoreceptive system that consists of the stalk and dorsal eyes, dermal photoreceptor cells, and photosensitive neurons. To examine the localization of photopigments, the stalk eyes were immunostained with specific antibodies to rhodopsin, retinochrome, and retinal-binding protein (RALBP), which had been generated against squid retinal proteins. The retina of the stalk eye was divided into villous, pigmented, somatic, and neural layers. It was comprised mainly of two types of visual and pigmented supportive cells. The type 1 visual (VC1) cell was characterized by well-developed microvilli on its apical protrusion and photic vesicles in the cytoplasm. The photic vesicles were specifically blackened by prolonged osmification. The type 2 visual (VC2) cell had less numerous, shorter microvilli on its concave apical surface and lacked photic vesicles. The anti-squid rhodopsin antiserum was localized specifically to the villous layer that corresponded to the VC1 microvilli. With the anti-retinochrome peptide antibody, the somatic layer showed specific but patchy, positive staining that corresponded to the cytoplasm of the VC1 cells. Because the photic vesicles are known to contain retinochrome, these results indicate that this retinochrome is localized in the VC1 cytoplasm. Anti-RALBP antibody stained the supranuclear cytoplasm to the distal cytoplasm of VC1 cells. This is the first demonstration of the localization of RALBP in the Gastropoda Onchidium stalk eye. In squid retina that were immunostained as positive controls, the anti-rhodopsin antibody stained rhabdomeric microvilli, the anti-retinochrome antibody stained the inner segment and the basal region of the outer segment, and the anti-RALBP antibody stained the outer and inner segments, respectively. These results suggest that the rhodopsin-retinochrome system that has been established in cephalopod eyes is present in the Onchidium stalk eye.

A role for lipid rafts in immune cell signaling.

Cross-linking of surface receptors in hematopoietic cells results in the enrichment of these receptors in the rafts along with other downstream signaling molecules. A possible explanation how signal is transduced through the plasma membrane has arisen from the concept of raft. From the study of cellular responses in the plasma membrane which enrich members of the Src-family tyrosine kinase, rafts can function as centers of signal transduction by forming patches. Under physiological conditions, these elements synergize to transduce successfully a signal at the plasma membrane. Rafts are suggested to be important in controlling appropriate protein interactions in hematopoietic cells, and aggregation of rafts following receptor ligation may be a general mechanism for promoting immune cell signaling.

Activation of the caspase cascade during Stx1-induced apoptosis in Burkitt's lymphoma cells.

Shiga toxin 1 (Stx1) produced by Escherichia coli has been reported to induce apoptosis in many different cell types, including Burkitt's lymphoma (BL) cells. Since it has been established that the caspases play essential roles as the effector molecules in the apoptotic process in most cases, we examined the kinetics of caspase activation during the process of Stx1-mediated apoptosis of BL cells. Using Ramos BL cells that are highly sensitive to Stx1-mediated cytotoxicity, we observed that multiple caspases, including caspase-3, -7, and -8 were promptly activated following Stx1 treatment, as indicated by both the procaspase cleavages and enhancement of cleavage of the tetrapeptide substrates of the caspases. In addition, the inhibition assay revealed that caspase-8 is located upstream of both caspase-3 and -7, suggesting that Stx1-mediated apoptosis utilizes a similar caspase cascade to that involved in Fas-mediated apoptosis. Neither anti-Fas mAb nor TNF-alpha, however, affected the Stx1-mediated apoptosis of Ramos cells. Although the precise mechanism of Stx1-mediated activation of caspase-8 is still unclear, we have demonstrated that crosslinkage of CD77, a functional receptor for Stx1, with specific antibody is sufficient to induce activation of caspase-8. Our findings should provide new insight into the understanding of the molecular basis of Stx1-mediated cell injury.

The Wilms' tumor gene product WT1 mediates the down-regulation of the rat epidermal growth factor receptor by nerve growth factor in PC12 cells.

Recently, we characterized the rat epidermal growth factor receptor (EGFR) promoter and demonstrated that TCC repeat sequences are required for the down-regulation of EGFR by nerve growth factor (NGF) in PC12 cells. In this study, we report that the Wilms' tumor gene product WT1, a zinc finger transcription factor, is able to enhance the activity of the rat EGFR promoter in cotransfection assays. Gel mobility shift assays demonstrate that WT1 binds to the TCC repeat sequences of the rat EGFR promoter. Overexpression of WT1 resulted in up-regulation of the expression levels of endogenous EGFR in PC12 cells. Interestingly, NGF down-regulated the expression levels of WT1 and EGFR in PC12 cells, but not in the p140(trk)-deficient variant PC12nnr5 cells or in cells expressing either dominant-negative Ras or dominant-negative Src. Most importantly, we evaluated the inhibitory effect of antisense WT1 RNA on EGFR expression, and we found that antisense WT1 RNA could substantially reduce EGFR repression in either histochemical staining study or immunoblot analysis. These results indicate that NGF-induced down-regulation of the EGFR in PC12 cells is mediated through WT1 and that WT1 may play an important role in the differentiation of nerve cells.

Activation of isoflavone biosynthesis in excised cotyledons of Lupinus seedlings by jasmonoids and excess light.

Exogenous jasmonic acid (JA) and methyl jasmonate (MJ) induced accumulation of isoflavone constituents in cotyledons prepared from imbibed seeds of white lupin (Lupinus albus L.). Exogenous 0.2 mM MJ enhanced the levels of 7-O-(6"-O-malonyl)glucosylgenistein and 7-O-glucosylgenistein in the cotyledons of etiolated seedlings that had been incubated in the dark for 48 h. Regarding isoflavone induced by excision and slicing in the cotyledons as background level, the effect of light was 2- to 3-fold higher than that of 0.2 mM MJ. Cotyledons exposed to MJ along with a 24-h light period displayed a higher level of isoflavone accumulation than that of light alone. Total molar amounts of isoflavone accumulated in the cotyledons treated with MJ under continuous light were approximately the sum of those induced by MJ alone and light alone, respectively. The additive-like effect of MJ and light on isoflavone accumulation in lupin tissues suggested the presence of two different signaling systems independently responsible for those two stimuli. Excised cotyledons from etiolated yellow lupin (L. luteus L. cv. Topaz) seedlings also supported this hypothesis. The cotyledons could accumulate both an isoflavone and a flavone, and MJ selectively increased some of the isoflavone constituents, whereas light enhanced the levels of both. The selective accumulation mechanism of isoflavonoids in cotyledons, in which jasmonoids are involved, clearly differed from that activated by light.

A double cysteine trkA mutant exhibiting reduced NGF binding and delayed Erk signaling.

The NGF receptor trkA is a tyrosine kinase receptor comprising an extracellular domain with a ligand-binding site, a transmembrane-spanning domain (TMD), and an intracellular domain composed of a juxtamembrane region (JMR), a tyrosine kinase domain, and a short carboxy-terminal tail. Nerve growth factor (NGF) binds and activates this receptor, leading to phosphorylation of signaling substrates involved in neuronal proliferation, differentiation, and survival. Human trkA contains one cysteine residue in the TMD (C423) and another, separated by 12 residues, in the JMR (C436). We hypothesized that the removal of one or both of the cysteines would affect NGF-induced signaling of the trkA receptor. Here we show that NGF induces rapid receptor autophosphorylation in a wild-type, trkA-expressing clone (WT11), in a single cysteine trkA mutants (C423T or C436A), but lower autophosphorylation activity in a double-cysteine trkA mutant (C423T/C436A). WT11 and SM cells had similar binding affinity, but that of DM cells was lower, according to the NGF radioreceptor assay. NGF-induced Erk phosphorylation was rapid in WT11 and C423T cells, but delayed in C436A and C423T/C436A cells. NGF induced [3H]thymidine incorporation into WT11 and SM cells, but had no effect on DM cells. However, basic fibroblast growth factor (bFGF) induced rapid phosphorylation of Erk1/2, and [3H]thymidine incorporation in NIH3T3, WT11, single mutant (SM), and double mutant (DM) cells, suggesting that the impaired NGF-induced Erk phosphorylation and thymidine incorporation observed in DM cells are due to the double-cysteine mutations in the trkA receptor. Cumulatively, our findings support a model in which Cys436 of the trkA is responsible for the rapid transfer of the transmembrane occupancy signal to the SHC adaptor protein for activation of the Ras-Erk pathway and DNA synthesis.