The fidelity of base selection by the polymerase subunit of DNA polymerase III holoenzyme.

In common with other DNA polymerases, DNA polymerase III holoenzyme of E. coli selects the biologically correct base pair with remarkable accuracy. DNA polymerase III is particularly useful for mechanistic studies because the polymerase and editing activities reside on separate subunits. To investigate the biochemical mechanism for base insertion fidelity, we have used a gel electrophoresis assay to measure kinetic parameters for the incorporation of correct and incorrect nucleotides by the polymerase (alpha) subunit of DNA polymerase III. As judged by this assay, base selection contributes a factor of roughly 10(4)-10(5) to the overall fidelity of genome duplication. The accuracy of base selection is determined mainly by the differential KM of the enzyme for correct vs. incorrect deoxynucleoside triphosphate. The misinsertion of G opposite template A is relatively efficient, comparable to that found for G opposite T. Based on a variety of other work, the G:A pair may require a special correction mechanism, possibly because of a syn-anti pairing approximating Watson-Crick geometry. We suggest that precise recognition of the equivalent geometry of the Watson-Crick base pairs may be the most critical feature for base selection.     

Characterization of biological properties of synthetic and biological leukotriene B3

The effect of micropuncture of the renal papilla through an intact ureter on urinary concetrating ability of rats was examined. Micropuncture of the renal papilla caused a fall in urine osmolality in the punctured kidney from 1718 ± 106 to 1035 ± 79 mosmol/kg·H₂O. In order to investigate the role of renal prostaglandins in this process, PGE₂ excretion was measured and found to increase from 63.4 ± 14.0 to 205.5 ± 57.1 pg/min. Urine osmolality and PGE₂ excretion from the contralateral kidney were not significantly altered. In animals given meclofenamate (2 mg/kg·hr), renal PGE₂ excretion was reduced to 22.3 ± 5.1 pg/min prior to micropuncture and it remained low at 8.9 ± 1.8pg/min after papillary micropuncture. Meclofenamate also blocked the fall in urine osmolality caused by micropuncture of the renal papilla, with urine osmolality averaging 1940 ± 122 before and 1782 ± 96 mosmol/kg·H₂O after the micropuncture. These results indicated that papillary micropuncture through an intact ureter increased renal PGE₂ excretion and that a rise in renal production of PGE₂ or some other prostanoid is associated with a fall in urine concentrating ability.     

Assessment of Amyloid β Protein in Cerebrospinal Fluid as an Aid in the Diagnosis of Alzheimer's Disease

Abstract: The principal constituent of amyloid plaques found in the brains of individuals with Alzheimer's disease (AD) is a 39–42-amino-acid protein, amyloid β protein (Aβ). This study examined whether the measurement of Aβ levels in CSF has diagnostic value. There were 108 subjects enrolled in this prospective study: AD (n = 39), non-AD controls (dementing diseases/syndromes; n = 20), and other (n = 49). CSF was obtained by lumbar puncture, and Aβ concentrations were determined using a dual monoclonal antibody immunoradiometric sandwich assay. The mean Aβ value for the AD group (15.9 ± 6.8 ng/ml) was not significantly different from that for the non-AD control group (13.0 ± 7.1 ng/ml; p = 0.07), and substantial overlap in results were observed. Aβ values did not correlate with age ( r = −0.05, p = 0.59), severity of cognitive impairment ( r = 0.22, p = 0.21), or duration of AD symptoms ( r = 0.14, p = 0.45). These findings are in conflict with other reports in the literature; discrepant results could be due to the instability of Aβ in CSF. Aβ immunoreactivity decays rapidly under certain conditions, particularly multiple freeze/thaw cycles. Use of a stabilizing sample treatment buffer at the time of lumbar puncture allows storage of CSF without loss of Aβ reactivity. In conclusion, the total CSF Aβ level is not a useful marker for current diagnosis of AD.     

Herbivore-induced changes in plant carbon allocation: assessment of below-ground C fluxes using carbon-14

Effects of above-ground herbivory on short-term plant carbon allocation were studied using maize (Zea mays) and a generalist lubber grasshopper (Romalea guttata). We hypothesized that above-ground herbivory stimulates current net carbon assimilate allocation to below-ground components, such as roots, root exudation and root and soil respiration. Maize plants 24 days old were grazed (c. 25–50% leaf area removed) by caging grasshoppers around individual plants and 18 h later pulse-labelled with¹⁴CO₂. During the next 8 h,¹⁴C assimilates were traced to shoots, roots, root plus soil respiration, root exudates, rhizosphere soil, and bulk soil using carbon-14 techniques. Significant positive relationships were observed between herbivory and carbon allocated to roots, root exudates, and root and soil respiration, and a significant negative relationship between herbivory and carbon allocated to shoots. No relationship was observed between herbivory and¹⁴C recovered from soil. While herbivory increased root and soil respiration, the peak time for¹⁴CO₂ evolved as respiration was not altered, thereby suggesting that herbivory only increases the magnitude of respiration, not patterns of translocation through time. Although there was a trend for lower photosynthetic rates of grazed plants than photosynthetic rates of ungrazed plants, no significant differences were observed among grazed and ungrazed plants. We conclude that above-ground herbivory can increase plant carbon fluxes below ground (roots, root exudates, and rhizosphere respiration), thus increasing resources (e.g., root exudates) available to soil organisms, especially microbial populations.     

Homologs of the yeast neck filament associated genes: isolation and sequence analysis of Candida albicans CDC3 and CDC10

Morphogenesis in the yeast Saccharomyes cerevisiae consists primarily of bud formation. Certain cell division cycle (CDC) genes, CDC3, CDC10, CDC11, CDC12, are known to be involved in events critical to the pattern of bud growth and the completion of cytokinesis. Their products are associated with the formation of a ring of neck filaments that forms at the region of the mother cell-bud junction during mitosis. Morphogenesis in Candida albicans, a major fungal pathogen of humans, consists of both budding and the formation of hyphae. The latter is thought to be related to the pathogenesis and invasiveness of C. albicans. We have isolated and characterized C. albicans homologs of the S. cerevisiae CDC3 and CDC10 genes. Both C. albicans genes are capable of complementing defects in the respective S. cerevisiae genes. RNA analysis of one of the genes suggests that it is a regulated gene, with higher overall expression levels during the hyphal phase than in the yeast phase. Not surprisingly, DNA sequence analysis reveals that the proteins share extensive homology at the amino acid level with their respective S. cerevisiae counterparts. Related genes are also found in other species of Candida and, more importantly, in filamentous fungi such as Aspergillus nidulans and Neurospora crassa. A database search revealed significant sequence similarity with two peptides, one from Drosophila and one from mouse, suggesting strong evolutionary conservation of function.     

High-efficiency and multilocus targeted integration in CHO cells using CRISPR-mediated donor nicking and DNA repair inhibitors

Efforts to leverage clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) for targeted genomic modifications in mammalian cells are limited by low efficiencies and heterogeneous outcomes. To aid method optimization, we developed an all-in-one reporter system, including a novel superfolder orange fluorescent protein (sfOrange), to simultaneously quantify gene disruption, site-specific integration (SSI), and random integration (RI). SSI strategies that utilize different donor plasmid formats and Cas9 nuclease variants were evaluated for targeting accuracy and efficiency in Chinese hamster ovary cells. Double-cut and double-nick donor formats significantly improved targeting accuracy by 2.3–8.3-fold and 19–22-fold, respectively, compared to standard circular donors. Notably, Cas9-mediated donor linearization was associated with increased RI events, whereas donor nicking minimized RI without sacrificing SSI efficiency and avoided low-fidelity outcomes. A screen of 10 molecules that modulate the major mammalian DNA repair pathways identified two inhibitors that further enhance targeting accuracy and efficiency to achieve SSI in 25% of transfected cells without selection. The optimized methods integrated transgene expression cassettes with 96% efficiency at a single locus and with 53%–55% efficiency at two loci simultaneously in selected clones. The CRISPR-based tools and methods developed here could inform the use of CRISPR/Cas9 in mammalian cell lines, accelerate mammalian cell line engineering, and support advanced recombinant protein production applications.     

Changes in proteoglycan synthesis of chondrocytes in articular cartilage are associated with the time-dependent changes in their mechanical environment

Explant loading experiments were conducted to investigate the effect of load duration on proteoglycan synthesis. A compressive load of 0.1 MPa applied for 10 min was found to stimulate proteoglycan synthesis, while the same load applied for 20 h suppressed synthesis. This bimodal response suggests that the cells are responding to different mechanical stimuli as time progresses. A theoretical model has therefore been developed to describe the mechanical environment perceived by cells within soft hydrated tissues (e.g. articular cartilage) while the tissue is being loaded. The cells are modeled, using the biphasic theory, as fluid-solid inclusions embedded in and attached to a biphasic extracellular matrix of distinct material properties. A method of solution is developed which is valid for any axisymmetric loading configuration, provided that the cell radius, a, is small relative to the tissue height, h (i.e. h/a 1). A closed-form analytical solution for this inclusion problem is then presented for the confined compression configuration. Results from this model show that the mechanical environment in and around the cells is time dependent and inhomogeneous, and can be significantly influenced by differences in properties between the cell and the extracellular matrix.     

Intranuclear inclusions in the developing neurons of the rat cuneate nuclei

Summary The ultrastructure of intranuclear rodlets, microtubules, fibrillar lattices and membranous inclusions found in the developing cuneate nuclei of rats is described. Rodlets, ranging in diameter from 96–312 nm and in length from 1–2 m, are made up of tightly packed straight filaments measuring 5–8 nm in diameter. Microtubules with a diameter of 26 nm are clustered together. Fibrillar lattices are made up of fibrils with a diameter of 9 nm arranged in layers or sets. Two to nine sets make up a lattice, with a maximum width of 68 nm, in which the adjacent sets are arranged at an angle to each other. Rodlets and fibrillar lattices occur in 6.8% of the neurons. Membranous inclusions, reported here for the first time in normal neurons, are of 2 types: small vesicles of 0.1–0.6 m and large vacuoles measuring 1–2 m. Both types are bounded by either a single or a double membrane and generally have an electron lucent content. Membranous inclusions occur in 25.3 % of the neurons. Changes in the frequency of occurrence of the various intranuclear inclusions in the course of postnatal development are also reported.     

Development of a Defined Medium for Growth of Cercospora rosicola Passerini

A simple synthetic liquid medium containing a single amino acid, glucose, salts, trace metals, and thiamine was developed for cultivation of Cercospora rosicola Passerini. Thiamine was shown to be important to growth. Culture of C. rosicola Passerini in a chemically defined medium makes possible studies of (+)-abscisic acid biosynthesis and regulation.