Polarization splitting with cubic crystals evaluated with synchrotron radiation.

X-ray polarization-splitting crystals separate incident x rays into two components with perpendicular polarization by Bragg reflections at 45° from paired sets of internal planes. Here, the polarization-splitting properties of a germanium crystal are verified using incompletely polarized synchrotron radiation. Cleaner data would have come from a beam with a higher degree of polarization, which is achievable with small changes in the experimental geometry.

Extension of single-crystal x-ray spectropolarimetry with cubic crystals beyond perfect polarization-splitting geometries.

The single-crystal spectropolarimeter envisioned by Baronova and Stepanenko splits an incident x-ray beam into two beams with mutually orthogonal linear polarizations by using simultaneous reflections at the perfectly polarizing 45° Bragg angle on certain pairs of internal planes in hexagonal or cubic crystals. These planes intersect along a threefold symmetry axis, making a 120° angle with each other, and are typically symmetric with respect to the crystal surface. In practice, the wavelength of the diagnostic x-ray lines does not exactly satisfy Bragg's law for the crystal in the ideal polarizing orientation, so the extinction of reflections is incomplete. Accepting this limitation, this paper shows that for cubic crystals, other pairs of internal planes exist that satisfy the polarization requirements approximately. Typically, they are accessible from the perfect polarization-splitting geometry by small rotations of the crystal. This paper includes examples of such planes for cubic crystals with {110} and {211} surface cuts.

Cubic crystals in an x-ray polarization-splitting geometry.

Hexagonal and cubic crystals contain paired sets of internal planes that reflect the linearly polarized components of certain x rays into two separate, perpendicular directions. For the cubic crystals, two distinct crystal orientations provide the same polarization-splitting geometry. One of the orientations may have advantages for plasma spectroscopy by suppressing unwanted reflections. This paper demonstrates the two orientations with a germanium crystal and K characteristic lines from copper and zirconium.

Proliferation studies on chromosome preparations of bone marrow in hematological disease.

The growth rate of neoplastic cells has been the subject of numerous scientific and diagnostic approaches. The study presented here analyses the relationship between mitotic activity in standardised cytogenetic bone marrow preparations from three haematological diseases and diagnostic and clinical parameters, most importantly the outcome. The disorders studied were: Acute lymphoblastic leukemia (ALL) (N=107), chronic myeloid leukemia (CML) (N=166) and aplastic anemia in childhood (AA) (N=39). A strict protocol of quantitative standardisation of cytogenetic slides was adhered to ensuring comparability both cross-sectionally and longitudinally. The samples were studied after short-term incubation without mitogenic in vitro stimuli. The most important findings include: i) ALL: Immunological subtypes can be differentiated according to their proliferation profile; there is a striking difference between childhood and adult ALL in proliferation activity; most importantly initial proliferation is much higher in patients who will relapse than in those with stable remission. ii) CML: Philadelphia-positive CML shows proliferation activities quite distinct from Philadelphia-negative CML; however there is only a small change in the proliferative activity from the chronic phase to the accelerated phase or blast crisis. iii) AA: Very low proliferation scores rise quickly to near normal levels during immunosuppressive therapy in most patients. Higher levels at diagnosis are associated with a faster and better response to therapy. In conclusion, assessment of the proliferative activity in cytogenetic preparations made from bone marrow samples of patients with haematological disease may add valuable information as to diagnostic sub-groups and clinical course and may contribute to therapeutic decisions.

Preparation, structure, and properties of a new giant manganese oxo-alkoxide wheel, [Mn19O12(OC2H4OCH3)14(HOC2H4OCH3)10]. HOC2H4OCH3.

Alkoxides are of great interest as precursors for sol-gel processing of advanced ceramic materials, but there is very little general knowledge about the low-valent 3d-element alkoxides. The novel oxo-alkoxide, [Mn19O12(moe)14-(moeH)10].MOEH (MOE = OC2H2-OCH3), was prepared, by metathesis and auto-decomposition, from MnCl2 and potassium methoxyethoxide in toluene/MOEH, and the solid-state structure was determined from single-crystal X-ray diffraction data: trigonal cell, space group R3 (no. 148), a = 27.560(3), c = 19.294(2) A, Z = 3, R1 = 0.0737, wR2 = 0.1609. The individual molecules are shaped as flat discs and all Mn atoms are divalent and octahedrally coordinated by oxygen atoms in a CdI2-type layer structure. The central Mn atom is coordinated by six mu3-oxo atoms, the six middle ring Mn-atoms by two mu3-oxo atoms and four MOE(H) groups, while the peripheral ring contains twelve Mn atoms coordinated by one mu3-oxo atom and five MOE(H) groups. Differential scanning calorimetry studies showed that the first and irreversible changes start at about 100 degrees C. The continuous decrease of (chiM)T with decreasing temperature below 150 K in the magnetic susceptibility measurements is probably due to antiferromagnetic interactions. FTIR and UV/Vis spectroscopy of solid and dissolved samples showed that the solid-state structure changes at least to some extent on dissolution in toluene/MOEH.

Permanent embryonic germ cell lines of BALB/cJ mice--an in vitro alternative for in vivo germ cell mutagenicity tests.

To offer a sensitive and predictive in vitro method to assess germ cell mutagenicity, we established primordial germ (PG) cell-derived permanent female and male embryonic germ (EG) cell lines of the mouse (strain BALB/cJ). The differences in developmental sensitivity of EG cells and differentiated fibroblast cells of the mouse cell line 3T3 to genotoxicants were tested comparatively under identical test conditions. Cytotoxicity assay was measured by the MTT test and genotoxic effects were determined by sister chromatid exchanges (SCE) rates induced by standard reference mutagens. Both methods are used to assign the chemicals to two classes of in vivo reproductive toxicity, non- and strongly genotoxic to germ cells. Applying linear discriminant analysis, a biostatistical prediction model (PM) was developed for the female cell line EG(3). This procedure identified a single variable, the Ig(SCE(200)EG(3)) as the statistically significant concentration related increase of 200% in the mean number of SCEs per metaphase spread after 3 h of exposure to be sufficient for separation into the classes: non- and strongly genotoxic to germ cells. Applying this PM to the training set of five genotoxic and three non-genotoxic test chemicals, 100% correct classifications were obtained.

[Permanent embryonic mouse germ cell-lines, an in vitro alternative to in vivo germ cell mutagenicity tests]. / Permanente embryonale Keimzelllinien - eine in vitro Alternative zur Beurteilung von Keimzellmutagenität.

Germ cell mutagenesis is required by the 7th amendment of the directive 67/548 EEC into the national regulations on existing chemicals. Officially accepted in vivo test systems for stage specific mutagenicity are the dominant lethal (DL) test and the specific locus test (SLT) in mice. An acceptable in vitro alternative designed to address germ cell mutagenesis and discriminate between male and female specific effects is not available at present. In order to offer a sensitive and predictive in vitro method to assess the genotoxic potential of chemical agents on male and female reproduction, we established primordial germ (PG) cell-derived permanent embryonic germ (EG) cell lines of the mouse (strain BALB/cJ). The differences in developmental sensitivity of the EG(3) cell line and differentiated fibroblast cells 3T3 were comparatively tested with cytotoxicity assay (MTT test ) and genotoxic studies (SCE-assay) under identical test conditions. The concentration-response curves reflected the female cell line EG(3) to be extremely sensitive concerning cytotoxic and genotoxic endpoints. Therefore this cell line was used to classify in vivo genotoxic and non-genotoxic test substances with different potential endpoints. Applying linear discriminant analysis three endpoints were identified for the correct classification (100%) of all test chemicals, namely the SCE(200) value (increase of 200% in the mean number of SCEs per metaphase spread) for EG(3) (3 hrs and 24 hrs assay) and the IC(5)0 value for EG(3) after 3 hrs of exposure to test chemicals.

Embryotoxicity screening using embryonic stem cells in vitro: correlation to in vivo teratogenicity.

Blastocyst-derived pluripotent embryonic stem (ES) cells of the mouse can be induced to differentiate in culture into a variety of cell types, including cardiac muscle cells. The embryonic stem cell test that makes use of the differentiation of ES cells into cardiomyocytes in a standardized in vitro model was developed to offer an alternative method to comprehensive in vivo studies in reproductive toxicology about toxic effects of chemicals. ES cells of the mouse cell line D3 are investigated for their preserved capability to differentiate following drug exposure, and both ES cells and differentiated fibroblast cells of the mouse cell line 3T3 are comparatively analyzed for effects on viability. The following endpoints are used to classify the embryotoxic potential of chemicals into three classes of in vitro embryotoxicity (non-, weakly or strongly embryotoxic). These endpoints are: (1) the inhibition of differentiation of ES cells into cardiomyocytes after 10 days of treatment, and the decrease of viability (cytotoxicity) of (2) 3T3 cells and (3) ES cells after 10 days of treatment, determined by a 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) test. 50% inhibition concentrations for differentiation (ID(50)) and cytotoxicity (IC(50)D3 and IC(50)3T3) are calculated from concentration-response curves. Applying linear analysis of discriminance, a biostatistical prediction model (PM) was developed. This procedure identified three variables, the lg(IC(50)D3), the lg(IC(50)3T3) and the relative distance between IC(50)3T3 and ID(50), that improved the separation of the three classes of embryotoxicity compared to the prediction model that was originally proposed after test development. Unlike the original PM, the improved PM incorporates as one variable the relative distance between IC(50)3T3 and ID(50), instead of the ratio ID(50)/IC(50)D3 that was used previously.

Prevalidation of the Embryonic Stem Cell Test (EST)-A New In Vitro Embryotoxicity Test.

Pluripotent embryonic stem cells (ES cells) of the mouse (cell-line D3) can be maintained in the undifferentiated state in the presence of LIF (Leukaemia Inhibitory Factor). Upon withdrawal of LIF, these cells differentiate into various cell types under appropriate conditions. This property of ES cells allowed us to develop an in vitro embryotoxicity test, the Embryonic Stem Cell Test (EST; In Vitro Toxicology 1997, 10, 119-127), which does not require taking embryonic cells or tissues from pregnant animals. In the EST, the effect of test chemicals on three endpoints is assessed: inhibition of the differentiation of ES cells into contracting myocard, cytotoxicity in ES cells and cytotoxicity in mouse 3T3 fibroblasts, which are serving as differentiated cells in the test. The results of a prevalidation study of the EST are described, which was conducted according to the ECVAM prevalidation scheme. In the first stage of the study (Phase I), a standard operating procedure (SOP) was elaborated. In the second phase (Phase II), the interlaboratory transferability of the EST was assessed using three test chemicals representing three classes of embryotoxicity (a strong, a weak and a non-embryotoxic chemical) in two European laboratories (ZEBET at the BgVV in Berlin, Germany; ECVAM at the JRC in Ispra, Italy) and one US laboratory (Institute for In Vitro Sciences (IIVS) in Gaithersburgh, MA, USA). In the final stage of prevalidation (Phase III), nine test chemicals and a positive control were tested under blind conditions at ZEBET and ECVAM. The statistical evaluation of the results led to the development of an improved prediction model for the EST.

An in vitro embryotoxicity assay using the differentiation of embryonic mouse stem cells into haematopoietic cells.

The earliest identified state of a specific cell in mammalian embryonic development is the embryonic stem cell (ES cell). The murine ES cell line D3 was used to establish conditions that allow a reproducible differentiation of ES cells in culture. The development of haematopoietic cells in semi-solid medium parallels to some extent the onset of haematopoiesis in the developing embryo. In methylcellulose cultures, erythropoiesis is observed at day 7 to day 8 of culture. An inhibitory influence of retinoic acid is observed both on blood cell development and on myocardial cell development. In contrast, retinoic acid induces the development of nerve and skeletal muscle cell differentiation in D3 ES cells. Further optimization of the culture conditions for ES cell differentiation will facilitate the use of ES cells for embryotoxicity testing in vitro.

Development of an in vitro embryotoxicity test using murine embryonic stem cell cultures.

Mouse embryonic stem (ES) cell line D3 was used to establish conditions for a reproducible differentiation of ES cells in culture. ES cells can be maintained in an undifferentiated state by cultivation on a feeder layer of embryonic fibroblasts. ES cells form aggregates in suspension and can spontaneously differentiate into complex organized embryoid bodies (EBs), which in many respects resemble early postimplantation mouse embryos. Under appropriate culture conditions various cell and tissue types will develop in EBs: these include myocardial and skeletal muscle, nerve cells, chondrocytes and blood cells. Retinoic acid (RA) was used as an embryotoxic substance to test the application of ES cell cultures in in vitro embryotoxicity testing. RA (1 x 10(-8)m) induced an increase in skeletal muscle cell differentiation, which followed a characteristic pattern: day 10 is characterized by the first appearance of mononucleated myoblasts; day 12 shows the fusion of myoblasts; on day 13, multinucleated myotubes can be detected, and on day 25 contractile myofibres are present in ES cell cultures. The development of blood islands with red cells enhanced by erythropoietin in EBs has encouraged the hope that, subsequently, more mature stages of erythroid, myeloid and lymphoid cell development could occur in vitro. These data provide further support for the use of ES cells in an in vitro assay for embryotoxicity testing.

A versatile phage lambda expression vector system for cloning in Escherichia coli.

By integrating fragments from the expression plasmids pJK2 and pJK4 into a derivative of the bacteriophage lambda, we constructed the phage expression vectors lambda JK2 and lambda JK4, which allow efficient cloning of genomic or cDNA either into the 5' end or the 3' end of the lacZ gene of Escherichia coli. Expression of barrier-free DNA in phase may lead to fusion proteins consisting of active beta-galactosidase (beta Gal) plus an additional polypeptide encoded by the inserted DNA. Analysis of distinct recombinant clones is quick and easy, due to the reversible integration of the plasmid into the genome. As an example, we constructed an expression library of genomic Plasmodium falciparum DNA in lambda JK2. We polymerised (amplified) and expressed a synthetic DNA fragment, which codes for a potential antigenic determinant of the 11-1 gene of Plasmodium falciparum as a fusion to the N terminus of active beta Gal. We demonstrate that such chimeric molecules can be affinity-purified and that polypeptides can be separated from the beta Gal part by cleavage with the protease factor Xa.

Bone volume and mineral density of iliac crest spongiosa in uremia.

Bone mass and bone mineral content were measured in non-dialyzed and dialyzed uremic patients. Bone mass, measured by micromorphometry and a gas displacement method, was higher in uremic than in age and sex matched control subjects (micromorphometry-U:25.8 +/- 8.24%; Co:15.6 +/- 4.38; gas displacement-U:211 +/- 66 mm3/cm3; Co:191 +/- 45). In hemodialyzed patients, bone mass was lower the longer the patients had been on dialysis (r = 0.38; P 0.05). Bone mineral content (specific weight) was diminished in uremia (1.82 +/- 0.095 g/ml; controls 1.854 +/- 0.0173). In hemodialyzed patients, specific weight was higher, as was Ca content of bone assessed by neutron activation analysis. It is concluded, that negative Ca balance was the major cause of bone loss and that bone loss is thus preventable.