Attachment of blastocysts to lens capsule: A model system for trophoblast-epithelial cell interaction on a natural basement membrane

Summary The bovine lens capsule has previously been shown to provide an optimal surface for the examination of epithelial cell interaction with a basement membrane. This native substrate has been used to investigate some initial aspects of attachment of mouse blastocysts and trophoblastic cellular outgrowth. Mouse blastocysts were presented to the cell-free humoral side of the anterior lens capsule, incubated for 72 h, and examined by scanning and transmission electron microscopy. Blastocysts hatch and attach from their zonae pellucidae by 30 h. Trophoblastic cells proliferate rapidly in a coronal direction, display extensive surface microvilli, and advance by the extension of numerous filipodia, many of which terminate with bulbous projections. These projections were shown by transmission electron microscopy to contain numerous vacuoles and polysomes. To simulate further the initial blastocyst-uterine interaction, a suspension of lens epithelial cells was introduced to the capsule and permitted to form a monolayer prior to the addition of the blastocysts. At 72 h the monolayer of lens cells remained intact. We observed that: a) lens cells appear to recede from the advancing trophoblastic cells, and b) trophoblastic cells extend beneath the monolayer of lens cells and thereby dislodge the cells from the lens capsule substrate. No infiltration of the capsule by the advancing trophoblastic cells was observed. The lens capsule appears to offer a promising system for the study of trophoblast-epithelial cell interaction on a natural basement membrane.     

Effects of local density and flower colour polymorphism on pollination and reproduction in the rewardless orchid Dactylorhiza sambucina (L.) Soò

The rewardless orchid Dactylorhiza sambucina shows a stable flower colour polymorphism, with both yellow- and red-flowered morphs growing sympatrically. Pollination biology and breeding system were investigated to examine the effects of density of plants, colour polymorphism, inflorescence dimension, and flower position within inflorescence on male and female reproductive success in three natural populations of D. sambucina. There were significant differences among sites in the number of pollinia removed and in fruit set per inflorescence. Number of removed pollinia and capsule production in D. sambucina were independent from flower and inflorescence size or flower position. As a whole, the red morphs showed the highest number of capsules produced, while the yellow morphs had the greatest male success. The relative male and female reproductive success were independent from plant density but were significantly correlated with the yellow morph frequency at the population level. Overall, our findings show that the contribution to the total reproductive success deriving from the two colour morphs does not conform with the predictions of negative frequency-dependent selection.     

犬脑内动脉构筑的显微X线解剖学研究

犬脑11只,经生理盐水冲洗脑血管后,注入20％钡胶液,切成0.2～1.0厘米的厚片,用显微X线法研究犬脑内各级动脉的构筑,其结果:1.皮质动脉的管径平均为25.9±0.005微米,平均长度为888.0±0.241微米。其形态与发出部位有关,分别呈栅状和瓶刷状。2.髓质动脉的管径平均为49.9±0.007微米。呈直线或孤形向心走行。3.皮质下动脉的管径平均为38.7±0.009微米。呈新月形或蟹钳状分布。4.豆纹动脉和内囊动脉的平均管径为70.0±0.021微米。呈锐角、反血流方向发自母干,再呈“S”形上升。5.丘脑动脉的平均管径为63.7±0.019微米,主要从下方进丘脑,呈树枝状分支。     

Buried seed population in the herbaceous lomas on Loma Ancon in the coastal desert of central Peru

Herbaceous lomas in the Peruvian coastal desert, of South America establish in spring, and its habitat is limited to the southern or southwestern slopes along the coast that are affected by thick fog. The time of appearance, the duration and the thickness of the fog vary greatly from year to year, so the lomas can grow only in habitats with enough water to, sustain seed germination and plant growth. This paper studies the species composition and density of the buried seed population, of the herbaceous lomas of Loma Ancon in order to clarify the mechanisms of the lomas' establishment. The mean number of species with viable seeds was about, 12 spp. m⁻² and that of dead seeds was about 22 spp. m⁻². The dominant species wereSolanum tuberiferum, S. pinnatifidum andNolana humifusa, both in viable and dead seeds. Viable seed density was about 5000–8000 seeds m⁻², which is comparable with the seed densities of other herbaceous communities. Dead seed density was about 15000–27000 seeds m⁻², or nearly three times the viable seed density, because the rate of decomposition was slow in the extremely dry conditions. The net increase of viable seeds by seed production was estimated at about 5000 seeds m⁻² in 1980, and the increase in the number of dead seeds was 2200 seeds m⁻².     

Induced reversion of a Chinese hamster ovary triple auxotroph. Validation of the system with several mutagens

A Chinese hamster ovary triple auxotroph (CHO AUXB1) requires glycine, adenosine, and thymidine (GAT) for growth and survival due to a defect in the structural gene for folylpolyglutamate synthetase (FPGS). This auxotroph and others like it contain less than 3% of the parental amounts of FPGS activity. In order to develop a reverse mutation assay with CHO AUXB1, we determined the optimal conditions for measuring reversion and characterized some of the revertants. We also obtained quantitative mutagenicity data for several direct-acting mutagens for comparison to the parental CHO-S/HGPRT locus. Induced revertants appear in the culture immediately following 20-22 h exposures in +GAT complete medium, indicative of dominant genetic changes. They are maximally expressed after 2 population doublings and can be conveniently selected after 44-48 h of expression growth by plating 1 X 10(6) cells/100-mm dish into -GAT-deficient medium and incubating 12-13 days. Plating reconstruction experiments show that the cloning efficiencies of revertants in -GAT medium are not influenced by the presence of up to 1 X 10(6) CHO AUXB1 cells. Dose-dependent increases above the spontaneous revertant frequency (average = 5 X 10(7)) are induced with cis-Pt(NH3)2Cl2 (14-fold) (but not trans-Pt(NH3)2Cl2), PtCl4(10-fold), Pt(SO4)2 (14-fold), K2CrO4 (8-fold), EMS (10-fold), 4-NQO (53-fold), ICR-191 (60-fold), and ICR-170 (30-fold). All of the revertants that have been isolated are stable to repeated subculturing in -GAT medium; 40 out of 42 that have been analyzed are characterized by an increased 72-h growth incorporation of labeled folate and their extracts contain 5-94% as much FPGS as the original, parental CHO-S line. Spontaneous and induced reversion to the GAT+ phenotype primarily reflects mutations involving the FPGS gene locus. But the re-acquisition by most of the revertants of much less than normal amounts of FPGS activity suggests that they arise from compensatory second-site mutations within this gene. Comparison of the mutagenicity patterns of the foregoing compounds as a function of the applied concentration and the relative percent survival reveals some interesting similarities, as well as differences, between the CHO AUXB1/FPGS and CHO-S/HGPRT loci. In particular, the FPGS locus is rather insensitive to EMS (or other simple alkylating agents). However, it seems to be quite susceptible to reversion by other chemicals that are known to react selectively with guanine bases in DNA. CHO AUXBI is a useful supplemental mammalian assay system for assessing quantitatively the generally weak mutagenic activities of metal compounds.     

Cellulose and xylans in the interface capsule in symbiotic cells of actinorhizae

Summary Enzyme-gold affinity labeling was used to show that in mature infected cells of actinorhizal symbioses the capsule on the plant host side of the symbiotic interface contained cellulose and xylans. Host species examined for cellulose wereAlnus rubra, Casuarina equisetifolia, C. glauca, Ceanothus cuneata, C. velutinus, Elaeagnus pungens, andMyrica cerifera.. Cellulose was in the capsule throughout the infected cell, implying that during development cellulose synthase was present in the host cell membrane component of the symbiotic interface. Any possible degradation of capsule cellulose by the microsymbiont was either incomplete or transient, because the polymer was present in mature infected cells. Cellulose labeling inCeanothus andElaeagnus was less consistent than in the other species. Dual labeled capsules inCasuarina glauca andAlnus rubra showed a similar distribution of xylans and cellulose. Cytochemical studies indicate that the capsule contains three major classes of cell wall polysaccharides: cellulose, hemicellulose (xylans), and pectins (shown previously). This suggests that the capsule is essentially a thin, internal, tubular plant cell wall.Abbreviations Au₅ Au₁₅ colloidal gold particles with mean diameter of 5 and 15 nm, respectively - CBHI cellobiohydrolase I - CBHII cellobiohydrolase II - PBS phosphate-buffered saline     

Enzyme IIBcellobiose of the phosphoenol-pyruvate-dependent phosphotransferase system of Escherichia coli: backbone assignment and secondary structure determined by three-dimensional NMR spectroscopy.

The assignment of backbone resonances and the secondary structure determination of the Cys 10 Ser mutant of enzyme IIBcellobiose of the Escherichia coli cellobiose-specific phosphoenol-pyruvate-dependent phosphotransferase system are presented. The backbone resonances were assigned using 4 triple resonance experiments, the HNCA and HN(CO)CA experiments, correlating backbone 1H, 15N, and 13C alpha resonances, and the HN(CA)CO and HNCO experiments, correlating backbone 1H,15N and 13CO resonances. Heteronuclear 1H-NOE 1H-15N single quantum coherence (15N-NOESY-HSQC) spectroscopy and heteronuclear 1H total correlation 1H-15N single quantum coherence (15N-TOCSY-HSQC) spectroscopy were used to resolve ambiguities arising from overlapping 13C alpha and 13CO frequencies and to check the assignments from the triple resonance experiments. This procedure, together with a 3-dimensional 1H alpha-13C alpha-13CO experiment (COCAH), yielded the assignment for all observed backbone resonances. The secondary structure was determined using information both from the deviation of observed 1H alpha and 13C alpha chemical shifts from their random coil values and 1H-NOE information from the 15N-NOESY-HSQC. These data show that enzyme IIBcellobiose consists of a 4-stranded parallel beta-sheet and 5 alpha-helices. In the wild-type enzyme IIBcellobiose, the catalytic residue appears to be located at the end of a beta-strand.     

HNCCH-TOCSY,a triple resonance experiment for the correlation of backbone 13Cα and 15N resonances with aliphatic side-chain proton resonances and for measuring vicinal 13CO, 1Hβ coupling constants in proteins

Summary A 3D triple resonance experiment has been designed to provide intraresidual and sequential correlations between amide nitrogens and -carbons in uniformly ¹³C¹⁵N-labeled proteins. In-phase ¹³C magnetization is transferred to the aliphatic side-chain protons via the side-chain carbons using a CC-TOCSY mixing sequence. Thus, the experiment alleviates the resonance assignment process by providing information about the amino acid type as well as establishing sequential connectivities. Leaving the carbonyl spins untouched throughout the transfer from ¹³C to ¹H leads to E.COSY-type cross peaks, from which the ³J_{H co} coupling constants can be evaluated. The pulse sequence is applied to oxidized Desulfovibrio vulgaris flavodoxin.     

Use of ATP to characterize biomass viability in freely suspended and immobilized cell bioreactors

This work describes investigations into the viability of cells growing on 3,4-dichloroaniline (34DCA). Two bioreactors are employed for microbial growth, a continuous stirred tank (CST) bioreactor with a 2-L working volume, and a three-phase air lift (TPAL) bioreactor with a 3-L working volume. Experiments have been performed at several dilution rates between 0.027 and 0.115 h(-1) in the CST bioreactor and between 0.111 and 0.500 h(-1) in the TPAL bioreactor. The specific ATP concentration was calculated at each dilution rate in the suspended biomass in both bioreactors as well as in the immobilized biomass in the TPAL bioreactor. The ATP was extracted from the cells using boiling tris-EDTA buffer (pH 7.75), and the quantity determined using a firefly (bioluminescence) technique. The cultures were inspected under an electron microscope to monitor compositional changes. Results from the CST bioreactor showed that the biomass-specific ATP concentration increases from 0.44 to 1.86 mg ATP g(-1) dry weight (dw) as dilution rate increases from 0.027 to 0.115 h(-1). At this upper dilution rate the cells were washed out. The specific ATP concentration reached a limiting average value of 1.73 mg ATP g(-1) dw, which is assumed to be the quantity of ATP in 100% viable biomass. In the TPAL bioreactor, the ATP level increased with dilution rate in both the immobilized and suspended biomass. The specific ATP concentration in the immobilized biomass increased from approximately 0.051 mg ATP g(-1) dw at dilution rates between 0.111 and 0.200 h(-1) to approximately 0.119 mg ATP g(-1) dw at dilution rates between 0.300 and 0.500 h(-1). This indicates that the immobilized biomass contained a viable cell fraction of around 5%. Based on these results, kinetic data for freely suspended cells should not be applied to the modeling of immobilized cell systems on the assumption that immobilized biomass is 100% viable. (c) 1993 John Wiley & Sons, Inc.     

Estimating tree-based dynamic treatment regimes using observational data with restricted treatment sequences

A dynamic treatment regime (DTR) is a sequence of decision rules that provide guidance on how to treat individuals based on their static and time-varying status. Existing observational data are often used to generate hypotheses about effective DTRs. A common challenge with observational data, however, is the need for analysts to consider “restrictions” on the treatment sequences. Such restrictions may be necessary for settings where (1) one or more treatment sequences that were offered to individuals when the data were collected are no longer considered viable in practice, (2) specific treatment sequences are no longer available, or (3) the scientific focus of the analysis concerns a specific type of treatment sequences (eg, “stepped-up” treatments). To address this challenge, we propose a restricted tree–based reinforcement learning (RT-RL) method that searches for an interpretable DTR with the maximum expected outcome, given a (set of) user-specified restriction(s), which specifies treatment options (at each stage) that ought not to be considered as part of the estimated tree-based DTR. In simulations, we evaluate the performance of RT-RL versus the standard approach of ignoring the partial data for individuals not following the (set of) restriction(s). The method is illustrated using an observational data set to estimate a two-stage stepped-up DTR for guiding the level of care placement for adolescents with substance use disorder.