Nanoscale Spin Manipulation with Pulsed Magnetic Gradient Fields from a Hard Disc Drive Writer.

The individual and coherent control of solid-state based electron spins is important covering fields from quantum information processing and quantum metrology to material research and medical imaging. Especially for the control of individual spins in nanoscale networks, the generation of strong, fast, and localized magnetic fields is crucial. Highly engineered devices that demonstrate most of the desired features are found in nanometer size magnetic writers of hard disk drives (HDD). Currently, however, their nanoscale operation in particular comes at the cost of excessive magnetic noise. Here, we present HDD writers as a tool for the efficient manipulation of single as well as multiple spins. We show that their tunable gradients of up to 100 µT/nm can be used to spectrally address individual spins on the nanoscale. Their gigahertz bandwidth allows one to switch control fields within nanoseconds, faster than characteristic time scales such as Rabi and Larmor periods, spin-spin couplings, or optical transitions, thus extending the set of feasible spin manipulations. We used the fields to drive spin transitions through nonadiabatic fast passages or to enable the optical readout of spin states in strong misaligned fields. Building on these techniques, we further apply the large magnetic field gradients for microwave selective addressing of single spins and show its use for the nanoscale optical colocalization of two emitters.

Sex selection of sperm in farm animals: status report and developmental prospects.

Pre-selection of spermatozoa based on the relative DNA difference between X- and Y-chromosome bearing populations by flow cytometry is an established method that has been introduced into commercial cattle production. Although several important improvements have increased the sort efficiency, the fertilising ability of sexed spermatozoa based on offspring per insemination is still behind farmers' expectations. The main stress factors, especially on mitochondria, that reduce the lifespan of spermatozoa are described, and new technical as well as biological solutions to maintain the natural sperm integrity and to increase the sorting efficiency are discussed. Among these methods are the identification of Y-chromosome bearing spermatozoa by bi-functionalised gold nanoparticles and triplex hybridisation in vivo as well as new laser-controlled deflection system that replaces the deflection of spermatozoa in the electrostatic field. Additionally, as well as a new nonsurgical transfer system of spermatozoa into the oviduct of cows has been developed and allows a significant reduction of spermatozoa per transfer. Altogether, the improvements made in the recent years will allow a broader use of sex-sorted spermatozoa even in those species that require more cells than cows and sheep.

Theoretical studies on magnetic circular dichroism by the finite perturbation method with relativistic corrections.

A theoretical method for calculating magnetic circular dichroism (MCD) of molecules is presented. We examined the numerical accuracy and the stability of the finite perturbation (FP) method and the sum-over-state (SOS) perturbation method. The relativistic effects are shown to be important for the MCD spectra of molecules containing heavy elements. Calculations using the FP and the SOS methods were carried out for ethylene, para- and ortho-benzoquinone, showing that the FP method is superior to the SOS method, as expected. The relativistic effect was examined using the second-order Douglas-Kroll Hamiltonians for the halogen molecules F2, Cl2, Br2, and I2. The Faraday terms of I2 and Br2 were strongly affected by the relativistic effects, while the effect was negligible for Cl2 and F2.

Peptides from the amino terminal mdm-2-binding domain of p53, designed from conformational analysis, are selectively cytotoxic to transformed cells.

We have synthesized three peptides from the mdm-2 binding domain of human p53, residues 12-26 (PPLSQETFSDLWKLL), residues 12-20, and 17-26. To enable transport of the peptides across the cell membrane and at the same time to maximize the active mdm-2 binding alpha-helical conformation for these peptides, each was attached at its carboxyl terminus to the penetratin sequence, KKWKMRRNQFWVKVQRG, that contains many positively charged residues that stabilize an alpha-helix when present on its carboxyl terminal end. All three peptides were cytotoxic to human cancer cells in culture, whereas a control, unrelated peptide attached to the same penetratin sequence had no effect on these cell lines. The same three cytotoxic peptides had no effect on the growth of normal cells, including human cord blood-derived stem cells. These peptides were as effective in causing cell death in p53-null cancer cells as in those having mutant or normal p53. Peptide-induced cell death is not accompanied by expression of apoptosis-associated proteins such as Bax and waf(p21). Based on these findings, we conclude that the antiproliferative effects of these p53-derived peptides are not completely dependent on p53 activity and may prove useful as general anticancer agents.

Cleavage of thyroxine-binding globulin during cardiopulmonary bypass.

Thyroxine-binding globulin (TBG) is a noninhibitory member of the serine protease inhibitor (serpin) superfamily. A characteristic serpin cleavage product of TBG has been demonstrated in sera of septic patients. We find that a similar cleavage product appears in serum during the rapid decline of immunoassayable TBG and thyroxine (T(4)) that is associated with the inflammatory response to cardiopulmonary bypass (CPB). In vitro cleavage of TBG by the serine protease, neutrophil elastase induces a conformational change that has previously been shown to weaken affinity for T(4.) In vitro protease cleavage also decreases immunoassayable TBG, probably because the conformational change decreases the availability of the TBG epitopes to the measuring antibody. Thus, the rapid decrease in immunoassayable TBG concentration previously attributed to accelerated clearance is caused in part by the proteolytic cleavage per se. The evidence for proteolysis of TBG concurrent with the decrease in serum T(4) during CPB is consistent with the proposed release of T(4) from TBG to cells showing serine protease activity.

Plasmid expression of a peptide that selectively blocks oncogenic ras-p21-induced oocyte maturation.

PURPOSE: We have previously found that a synthetic peptide corresponding to ras-p21 residues 96 110 (PNC2) selectively blocks oncogenic (Val 12-containing) ras-p21 protein-induced oocyte maturation. With a view to introducing this peptide into ras-transformed human cells to inhibit their proliferation, we synthesized an inducible plasmid that expressed this peptide sequence. Our purpose was to test this expression system in oocytes to determine if it was capable of causing selective inhibition of oncogenic ras-p21. METHODS: We injected this plasmid and a plasmid expressing a control peptide into oocytes either together with oncogenic p21 or in the presence of insulin (that induces maturation that is dependent on normal cellular ras-p21) in the presence and absence of the inducer isopropylthioglucose (IPTG). RESULTS: Microinjection of this plasmid into oocytes together with Val 12-p21 resulted in complete inhibition of maturation in the presence of inducer. Another plasmid encoding the sequence for the unrelated control peptide, X13, was unable to inhibit Val 12-p21-induced maturation. In contrast, PNC2 plasmid had no effect on the ability of insulin-activated normal cellular or wild-type ras-p21 to induce oocyte maturation, suggesting that it is selective for blocking the mitogenic effects of oncogenic (Val 12) ras p21. CONCLUSION: We conclude that the PNC2 plasmid selectively inhibits oncogenic ras-p21 and may therefore be highly effective in blocking proliferation of ras-induced cancer cells. Also, from the patterns of inhibition, by PNC2 and other ras- and raf-related peptides, of raf- and constitutively activated MEK-induced maturation, we conclude that PNC2 peptide inhibits oncogenic ras p21 downstream of raf.

Differences in patterns of activation of MAP kinases induced by oncogenic ras-p21 and insulin in oocytes.

Oncogenic ras (Val 12-containing)-p21 protein induces oocyte maturation by a pathway that is blocked by peptides from effector domains of ras-p21, i.e., residues 35-47 (that block Val 12-p21-activated raf) and 96-110 and 115-126, which do not affect the ability of insulin-activated cellular p21 to induce maturation. Oncogenic p21 binds directly to jun-N-terminal kinase (JNK), which is blocked by the p21 96-110 and 115-126 peptides. This finding predicts that oncogenic p21, but not insulin, induces maturation by early and sustained activation of JNK. We now directly confirm this prediction by showing that oncogenic p21 induces activating phosphorylation of JNK (JNK-P) and of ERK (MAP kinase) (MAPK-P), whose levels correlate with oocyte maturation. p21 peptides 35-47 and 96-110 block formation of JNK-P and MAPK-P, further confirming this correlation and suggesting, unexpectedly, that raf-MEK-MAPK and JNK-jun pathways strongly interact on the oncogenic p21 pathway. In contrast, insulin activates only low levels of JNK-P, and, surprisingly, we find that insulin induces only low levels of MAPK-P, indicating that insulin and activated normal p21 utilize MAP kinase-independent signal transduction pathways.

Partially bridge-fluorinated dimethyl bicyclo[1.1.1]pentane-1,3-dicarboxylates: preparation and NMR spectra.

Direct fluorination of dimethyl bicyclo[1.1.1]pentane-1,3-dicarboxylate, obtained from [1.1.1]propellane prepared by an improved synthetic procedure, furnished esters of 14 of the 15 possible bridge-fluorinated bicyclo[1.1.1]pentane-1,3-dicarboxylic acids, isolated by preparative GC. Calculated geometries reflect the substitution pattern in a regular fashion compatible with Bent's rules. Considerable additional strain is introduced into the bicyclo[1.1.1]pentane cage by polyfluorination; it is calculated to be as high as 33-35 kcal/mol for hexasubstitution. Three arrangements of the fluorine substituents are especially strain-rich: geminal, proximate, and W-related. The (1)H, (13)C, and (19)F NMR spectra exhibit a striking variety of chemical shifts and long-range coupling constants. These are in good agreement with results calculated with neglect of the bridgehead substituents for all of the chemical shifts by the GIAO-RHF/6-31G//RHF/6-31G and GIAO-RHF/6-31G//MP2/6-31G methods and for many of the coupling constants by the EOM-CCSD/6-311G//MP2/6-311G method. The proximate (4)J(FF) constants are particularly large (50-100 Hz) and show an inverse linear dependence on the calculated F-F distance in the range 2.43-2.58 A.

LiCB(11)Me(12): a catalyst for pericyclic rearrangements.

[structure: see text] Benzene and 1,2-dichloroethane solutions of the Li(+) salt of the weakly coordinating anion CB(11)Me(12)(-) catalyze the rearrangement of cubane to cuneane, quadricyclane to norbornadiene, basketene to Nenitzescu's hydrocarbon, and diademane to triquinacene. The Claisen rearrangement of phenyl allyl ether is also strongly accelerated.

Molecular dynamics of a grid-mounted molecular dipolar rotor in a rotating electric field.

Classical molecular dynamics is applied to the rotation of a dipolar molecular rotor mounted on a square grid and driven by rotating electric field E(nu) at T approximately 150 K. The rotor is a complex of Re with two substituted o-phenanthrolines, one positively and one negatively charged, attached to an axial position of Rh(2)(4+) in a [2]staffanedicarboxylate grid through 2-(3-cyanobicyclo[1.1.1]pent-1-yl)malonic dialdehyde. Four regimes are characterized by a, the average lag per turn: (i) synchronous (a < 1/e) at E(nu) = /E(nu)/ > E(c)(nu) [E(c)(nu) is the critical field strength], (ii) asynchronous (1/e < a < 1) at E(c)(nu) > E(nu) > E(bo)(nu) > kT/mu;, [E(bo)(nu) is the break-off field strength], (iii) random driven (a approximately 1) at E(bo)(nu) > E(nu) > kT/mu, and (iv) random thermal (a approximately 1) at kT/mu > E(nu). A fifth regime, (v) strongly hindered, W > kT, E(mu), (W is the rotational barrier), has not been examined. We find E(bo)(nu)/kVcm(-1) approximately (kT/(mu))/kVcm(-1) + 0.13(nu/GHz)(1.9) and E(c)(nu)/kVcm(-1) approximately (2.3kT/(mu))/kVcm(-1) + 0.87(nu/GHz)(1.6). For nu > 40 GHz, the rotor behaves as a macroscopic body with a friction constant proportional to frequency, eta/eVps approximately 1.14 nu/THz, and for nu < 20 GHz, it exhibits a uniquely molecular behavior.

Conformations of linear chains. Systematics and suggestions for nomenclature.

There are six categories of calculated favored dihedral angles in linear M(n)X(2n+2) chains. The Prelog-Klyne nomenclature is not helpful for classifying them, and we propose the following labels and symbols: A, anti, reserved for torsional angles within a few degrees of 180 degrees; T, transoid, omega approximately 165 degrees; D, deviant, omega approximately 150 degrees; O, ortho, omega approximately 90 degrees; G, gauche, omega approximately 60 degrees; C, cisoid, omega approximately 40 degrees. With the exception of C, all of these categories have been observed in alkanes, perfluoroalkanes, or oligosilanes.

Cytofluorometric analysis of the neutrophil-specific BH2-Ag on cells from neonates to adults.

Monoclonal antibody (MAB) BH2C6 recognizes a plasma membrane antigen, the BH2-Ag, specifically expressed by human neutrophils. While studies with peripheral blood and bone marrow from healthy adults clearly demonstrate the absence of BH2-Ag from other cellular components except neutrophils, they also indicate that the BH2-Ag is expressed more strongly by mature than immature neutrophils. The purpose of this study was to determine the expression of the BH2-Ag by peripheral blood neutrophils from premature newborns to adults. Seventy-two donors were studied in six age groups: newborns <36 weeks of gestational age; newborns >36 weeks of gestational age; 0.5-2 years; 4-8 years; 12-17 years; >30 years. Expression of the BH2-Ag by peripheral blood neutrophils was examined by cytofluorography using MAB BH2-C6 directly labeled with fluorescein isothiocyanate (FITC). Neutrophils were reacted in parallel with FITC-MAB directed against CD11b, the alpha-chain of the CD11b/CD18 antigen (CR3). BH2-Ag is expressed by 98.3-99.6% of the neutrophils in all groups, and is absent on other blood cells, including those of very premature newborns. Statistical comparisons with respect to the mean fluorescence intensity of the FITC-MAB BH2C6 bound did not support a significant difference in the expression of BH2-Ag in any age group. CD11b expression was also detected in every individual studied and its mean fluorescence intensity correlated significantly with that of BH2Ag (p <0.001). The uniform presence of BH2Ag in every individual including a very premature infant suggests that BH2-Ag is likely to be an essential component of neutrophil development in humans. A highly significant correlation between the mean fluorescence intensity obtained with MAB BH2C6 and MAB CD11b suggests a possible interactive role of the two antigens in neutrophil development and/or function.

A characteristic serpin cleavage product of thyroxine-binding globulin appears in sepsis sera.

T4-binding globulin (TBG), the principal thyroid hormone-binding protein of serum, is a member of the serine protease inhibitor (serpin) superfamily. We report a characteristic serpin cleavage product of TBG in sepsis sera. At 49-50 kDa, the TBG remnant is 4-5 kDa smaller than the intact protein and is the same molecular mass as a TBG cleavage product produced by incubation with polymorphonuclear elastase. Incubation with polymorphonuclear leukocytes also produces the 49- to 50-kDa remnant, and this proteolysis is stimulated by zymosan activation. Polymorphonuclear cell cleavage of TBG increases the ratio of free/bound T4. As previously described, in vitro cleavage of TBG by elastase also increases free/bound T4. These findings are consistent with the hypothesis that serine proteases present at inflammatory sites cleave TBG, releasing its hormonal ligands.

Expression of Fc(gamma)r1 (CD64) on polymorphonuclear leucocytes during progression to acquired immunodeficiency syndrome in perinatally human immunodeficiency virus-infected children.

CD64, the high-affinity receptor in the family of FCgamma receptors, is not expressed constitutively in polymorphonuclear leucocytes (PMN). CD64 is expressed by PMNs in the late stages of human immunodeficiency virus (HIV) infection in adults. We followed the expression of CD64 on PMNs in perinatally HIV-infected children during disease progression. Peripheral blood leucocytes (PBL) from 45 perinatally HIV-infected paediatric patients and 13 healthy age-matched controls were analysed using cytofluorimetry after reaction with a fluorophore-labelled monoclonal antibody (MoAb) to CD64. In parallel, we examined the expression of CD32, CD16, CD11b and the human neutrophil-specific BH2-Ag using fluorophore-labelled MoAbs. We found that up to 79.5% of the PMNs in children in class C3 express CD64. Most importantly, we observed a continuous and significant increase in the appearance of CD64+ PMNs as a function of CDC classification (P < 0.001) but no changes in the expression of CD32, CD16, CD11b and BH2-Ag. This suggests that following the expression of CD64 on PMNs can be useful in evaluating the progression of HIV infection in perinatally HIV-infected children.

Induction of oocyte maturation by jun-N-terminal kinase (JNK) on the oncogenic ras-p21 pathway is dependent on the raf-MEK signal transduction pathway.

PURPOSE: We have previously found that microinjection of activated MEK (mitogen activated kinase kinase) and ERK (mitogen-activated protein; MAP kinase) fails to induce oocyte maturation, but that maturation, induced by oncogenic ras-p21 and insulin-activated cell ras-p21, is blocked by peptides from the ras-binding domain of raf. We also found that jun kinase (JNK), on the stress-activated protein (SAP) pathway, which is critical to the oncogenic ras-p21 signal transduction pathway, is a strong inducer of oocyte maturation. Our purpose in this study was to determine the role of the raf-MEK-MAP kinase pathway in oocyte maturation and how it interacts with JNK from the SAP pathway. METHODS: We microinjected raf dominant negative mutant mRNA (DN-raf) and the MEK-specific phosphatase, MKP-T4, either together with oncogenic p21 or c-raf mRNA, into oocytes or into oocytes incubated with insulin to determine the effects of these raf-MEK-MAP kinase pathway inhibitors. RESULTS: We found that oocyte maturation induced by both oncogenic and activated normal p21 is inhibited by both DN-raf and by MKP-T4. The latter more strongly blocks the oncogenic pathway. Also an mRNA encoding a constitutively activated MEK strongly induces oocyte maturation that is not inhibited by DN-raf or by MKP-T4. Surprisingly, we found that oocyte maturation induced by JNK is blocked both by DN-raf and MKP-T4. Furthermore, we discovered that c-raf induces oocyte maturation that is inhibited by glutathione-S-transferase (GST), which we have found to be a potent and selective inhibitor of JNK. CONCLUSION: We conclude that there is a strong reciprocal interaction between the SAP pathway involving JNK and the raf-MEK-MAP kinase pathway and that oncogenic ras-p21 can be preferentially inhibited by MEK inhibitors. The results imply that blockade of both MEK and JNK-oncogenic ras-p21 interactions may constitute selective synergistic combination chemotherapy against oncogenic ras-induced tumors.

Selective binding of a monoclonal antibody to Aspergillus niger glucose oxidase by formaldehyde fixed human polymorphonuclear leukocytes.

BACKGROUND: Many of the procedures used in handling neutrophils may affect the expression of surface antigens, and hence their quantitation by flow cytometry. METHODS: Because the enzyme glucose oxidase of Aspergillus niger is absent in human tissues, an IgM against it (mAb GO) was used as negative control in a study involving the normal expression of neutrophil specific BH2-Ag in different age groups. RESULTS: When peripheral blood leukocytes (PBL) were freshly prepared, processed and stained with FITC-mAb GO without fixation or when the cells were stained with FITC-mAb GO prior to fixation with 2% formaldehyde, both median fluorescent intensity (MFI) and per cent of positively stained polymorphonuclear leukocytes (PMN) were similar to that obtained with a background sample without any antibody. However, when PBL were fixed after isolation with different concentrations of formaldehyde and for varying durations, MFI and per cent of positively stained PMN but not of monocytes or lymphocytes with FITC-mAb GO increased in a time and concentration dependent manner. Saturation was achieved at a finite concentration of the antibody. In a competition assay unlabelled mAb GO reduced binding of FITC-mAb GO to PMN by 79% and 95% at concentrations 100 and 200 times that of FITC labelled antibody, respectively. CONCLUSIONS: These observations strongly suggest that formaldehyde fixation causes the expression or accessibility of an epitope on PMN that is specifically recognized by the mAb GO.