Differential expression inArabidopsis ofLhca2, a PSI cab gene

A cDNA clone of the geneLhca2 encoding a photosystem I (PSI) type II chlorophylla/b-binding protein was isolated fromArabidopsis thaliana. The isolation of this, the fourth PSI cab gene fromArabidopsis, confirms a previous report [1] that indicatedArabidopsis may contain all four PSI cab genes identified in other plant species.Lhca2 is a single-copy gene as are the other knownArabidopsis PSI cab genes. The patterns of developmental expression and tissue-specific regulation ofLhca2 are similar to those of other PSI and PSII cab genes, but the light induction pattern and the steady-state mRNA level ofLhca2 are distinct. This suggests that a different mechanism may be employed to regulate the expression ofLhca2.     

Plant Effects on Spatial and Temporal Patterns of Nitrogen Cycling in Shortgrass Steppe

Because of the water-limited nature and discontinuous plant cover of shortgrass steppe, spatial patterns in ecosystem properties are influenced more by the presence or absence of plants than by plant type. However, plant type may influence temporal patterns of nutrient cycling between plant and soil. Plants having the carbon-3 (C₃) or carbon-4 (C₄) photosynthetic pathway differ in phenology as well as other attributes that affect nitrogen (N) cycling. We estimated net N mineralization rates and traced nitrogen-15 (¹⁵N) additions among plant and soil components during May, July, and September of 1995 in native plots of C₃ plants, C₄ plants, or mixtures of C₃ and C₄. Net N mineralization was significantly greater in C₃ plots than in C₄ plots during both July and September. C₃ plots retained significantly more ¹⁵N in May than did mixed and C₄ plots; these differences in ¹⁵N retention were due to greater ¹⁵N uptake by C₃ plants than by C₄ plants during May. There were no significant differences in total ¹⁵N retention among plant communities for July and September. Soil ¹⁵N was influenced more by presence or absence of plants than by type of plant; greater quantities of ¹⁵N remained in soil interspaces between plants than in soil directly under plants for July and September. Our results indicate that plant functional type (C₃ versus C₄) can affect both the spatial and the temporal patterns of N cycling in shortgrass steppe. Further research is necessary to determine how these intraseasonal differences translate to longer-term and coarser-scale effects of plants on N cycling, retention, and storage. Received 8 December 1997; accepted 6 May 1998.     

Selective enrichment and isolation of Rhodopseudomonas palustris using trans-cinnamic acid as sole carbon source

Abstract Enrichment cultures for anoxygenic phototrophs capable of using cinnamic acid as sole organic carbon source consistently yielded the nonsulfur purple bacterium Rhodopseudomonas palustris . Pure cultures of R. palustris obtained from the enrichments grew photoheterotrophically on cinnamate and benzoate as well as on derivatives of these compounds. Photosynthetic growth on cinnamate was greatly stimulated by addition of exogenous CO₂, and resulted in breakage of the aromatic nucleus. Growth yield studies suggested that cinnamate was converted by R. palustris to intermediates that can be quantitatively assimilated into cell material.     

Targeting conserved water molecules: Design of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine Hsp90 inhibitors using fragment-based screening and structure-based optimization

Inhibitors of the Hsp90 molecular chaperone are showing promise as anti-cancer agents. Here we describe a series of 4-aryl-5-cyanopyrrolo[2,3-d]pyrimidine ATP competitive Hsp90 inhibitors that were identified following structure-driven optimization of purine hits revealed by NMR based screening of a proprietary fragment library. Ligand-Hsp90 X-ray structures combined with molecular modeling led to the rational displacement of a conserved water molecule leading to enhanced affinity for Hsp90 as measured by fluorescence polarization, isothermal titration calorimetry and surface plasmon resonance assays. This displacement was achieved with a nitrile group, presenting an example of efficient gain in binding affinity with minimal increase in molecular weight. Some compounds in this chemical series inhibit the proliferation of human cancer cell lines in vitro and cause depletion of oncogenic Hsp90 client proteins and concomitant elevation of the co-chaperone Hsp70. In addition, one compound was demonstrated to be orally bioavailable in the mouse. This work demonstrates the power of structure-based design for the rapid evolution of potent Hsp90 inhibitors and the importance of considering conserved water molecules in drug design.     

Effect of luteal-phase support on endometrial microRNA expression following controlled ovarian stimulation

ABSTRACT: BACKGROUND: Studies suggested that microRNAs influence cellular activities in the uterus including cell differentiation and embryo implantation. In assisted reproduction cycles, luteal phase support, given to improve endometrial characteristics and to facilitate the implantation process, has been a standard practice. The effect of different types of luteal phase support using steroid hormones in relation to endometrial miRNA profiles during the peri-implantation period has not seen described. This study was designed to evaluate the expression of miRNAs during the luteal phase following controlled ovarian stimulation for IVF and the influence of different luteal phase support protocols on miRNA profiles. METHODS: The study was approved by the Johns Hopkins Hospital Institutional Review Board. Endometrial biopsies were obtained on the day of oocyte retrieval from 9 oocyte donors (group I). An additional endometrial biopsy was obtained 3-5 days later (Group II) after the donors were randomized into three groups. Group IIa had no luteal-phase support, group IIb had luteal support with micronized progesterone (P), and Group IIc had luteal support with progesterone plus 17-beta-estradiol (P+E). Total RNA was isolated and microarray analysis was performed using an Illumina miRNA expression panel. RESULTS: A total of 526 miRNAs were identified. Out of those, 216 miRNAs were differentially regulated (p<0.05) between the comparison groups. As compared to the day of retrieval, 19, 11 and 6 miRNAs were differentially regulated more than 2 fold in the groups of no support, in the P support only, and in the P+E support respectively, 3-5 days after retrieval. During the peri-implantation period (3-5 days after retrieval) the expression of 33 and 6 miRNAs increased, while the expression of 3 and 0 miRNAs decreased, in the P alone and in the P+E group respectively as compared to the no steroid supplementation group. CONCLUSION: Luteal support following COS has a profound influence on miRNA profiles. Up or down regulation of miRNAs after P or P+E support suggest a role(s) of luteal support in the peri-implantation uterus in IVF cycles through the regulation of associated target genes.     

Joint Variable Selection for Fixed and Random Effects in Linear Mixed‐Effects Models

Summary It is of great practical interest to simultaneously identify the important predictors that correspond to both the fixed and random effects components in a linear mixed‐effects (LME) model. Typical approaches perform selection separately on each of the fixed and random effect components. However, changing the structure of one set of effects can lead to different choices of variables for the other set of effects. We propose simultaneous selection of the fixed and random factors in an LME model using a modified Cholesky decomposition. Our method is based on a penalized joint log likelihood with an adaptive penalty for the selection and estimation of both the fixed and random effects. It performs model selection by allowing fixed effects or standard deviations of random effects to be exactly zero. A constrained expectation–maximization algorithm is then used to obtain the final estimates. It is further shown that the proposed penalized estimator enjoys the Oracle property, in that, asymptotically it performs as well as if the true model was known beforehand. We demonstrate the performance of our method based on a simulation study and a real data example.     

Respiratory distress and neonatal lethality in mice lacking Golgi alpha1,2-mannosidase IB involved in N-glycan maturation

There are three mammalian Golgi alpha1,2-mannosidases, encoded by different genes, that form Man5GlcNAc2 from Man(8-9)GlcNAc2 for the biosynthesis of hybrid and complex N-glycans. Northern blot analysis and in situ hybridization indicate that the three paralogs display distinct developmental and tissue-specific expression. The physiological role of Golgi alpha1,2-mannosidase IB was investigated by targeted gene ablation. The null mice have normal gross appearance at birth, but they display respiratory distress and die within a few hours. Histology of fetal lungs the day before birth indicate some delay in development, whereas neonatal lungs show extensive pulmonary hemorrhage in the alveolar region. No significant histopathological changes occur in other tissues. No remarkable ultrastructural differences are detected between wild type and null lungs. The membranes of a subset of bronchiolar epithelial cells are stained with lectins from Phaseolus vulgaris (leukoagglutinin and erythroagglutinin) and Datura stramonium in wild type lungs, but this staining disappears in lungs from null mice. Mass spectrometry of N-glycans from different tissues shows no significant changes in global N-glycans of null mice. Therefore, only a few glycoproteins required for normal lung function depend on alpha1,2-mannosidase IB for maturation. There are no apparent differences in the expression of several lung epithelial cell and endothelial cell markers between null and wild type mice. The alpha1,2-mannosidase IB null phenotype differs from phenotypes caused by ablation of other enzymes in N-glycan biosynthesis and from other mouse gene disruptions that affect pulmonary development and function.     

Enhanced transfection efficiency and improved cell survival after electroporation of G2/M-synchronized cells and treatment with sodium butyrate.

To achieve high transfection efficiency in human fibroblasts with good preservation of proliferative capacity we developed an electroporation procedure that combines two distinct modalities: use of recipient cells synchronized in the late G2/mitotic phase of the cell cycle and treatment of cells post-electroporation with 5 mM butyrate. This combination enabled reduction of plasmid DNA concentration and electroporation voltage, both associated with cytotoxicity, while greatly enhancing transfection efficiencies. Although the method was primarily developed for transient expression it was also found to improve stable expression. This procedure should have wide applicability, particularly in studies seeking to identify DNA sequences that lead to inhibition of DNA synthesis and proliferation in human fibroblasts and other cells refractory to transfection.     

cDNA cloning and expression of a potato (Solanum tuberosum) invertase

A cDNA clone encoding an invertase isoenzyme has been isolated from a potato leaf cDNA library. The deduced amino acid sequence shows significant similarities to previously characterised invertases. The highest degree of overall similarity, including the signal peptide sequence, is to carrot cell wall invertase, suggesting that the potato gene encodes an apoplastic enzyme. Expression of the gene, as determined by RT-PCR, is detected in stem and leaf tissue, and at lower levels in tuber, but is absent from roots.