Evidence for vitamin K semiquinone as the functional form of vitamin K in the liver vitamin K-dependent protein carboxylation reaction.

The protein carboxylating system derived from vitamin K-deficient rat liver microsomes functions in detergent solution if vitamin K₁, NADH, dithiothreitol, CO₂ and O₂ are added. The requirements for added NADH, dithiothreitol and O₂ are all eliminated by the use of vitamin K₁ hydroquinone in place of quinone. The use of the hydroquinone gives a more rapid reaction and a higher yield than does the quinone plus reducing system. The reaction proceeding from either the vitamin K₁ quinone or hydroquinone is blocked by the spin-trapping agent, 5,5-dimethyl-l-pyrroline-N-oxide, suggesting that the active form of vitamin K is the semiquinone.     

Developmental requirements of the univoltine species Chrysopa downesi: Photoperiodic stimuli and sensitive stages

Chrysopa downesi reproduces only in the spring and the resulting adults enter an aestival-autumnal-hibernal diapause which is primarily controlled by photoperiod. In the laboratory, constant photoperiods result in diapause induction and maintenance, whereas a series of short days followed by long days prevents or terminates diapause and promotes reproduction. The stages most sensitive to the diapause-averting stimulus are the free-living third instar, the third instar within the cocoon, and the pupa.C. downesi responds in different ways to three aspects of photoperiod: (a) an all-or-none response (diapause prevention or induction) to a sequence of two critical photoperiods, (b) an all-or-none response (diapause prevention or induction) to the difference between the long and short daylengths (a 4 hr difference is sufficient to avert diapause but a 2 hr difference is not), and (c) a quantitative response to the absolute duration of day (or night) length (after the short day requirement is fulfilled the rate of diapause termination is related to daylength).Differences and similarities in phenological adaptations and in photoperiodic responses of C. downesi, C. carnea, and C. harrisii reflect the degree of phylogenetic relationship between these closely related species.     

Genetic Heterozygosity in Unreplicated Bacteriophage λ Recombinants

Bacteriophage crosses using density-labeled parents have been carried out under conditions restricting DNA synthesis. The parental material and genetic contributions to progeny manifesting recombination within a genetic interval sufficiently short to exhibit high negative interference have been examined. The unreplicated products of recombination isolated as phage particles appear to contain long continuous heteroduplex regions which are heterozygous for the closely linked markers. Recombination between closely linked markers seems to be the consequence of the removal of base-pair mismatches that are present within the heteroduplex regions. This localized reduction of heterozygosity within the heteroduplex regions that join the parental components of recombinant DNA molecules can account for high negative interference.     

Selective precipitation and purification of monovalent proteins using oligovalent ligands and ammonium sulfate

This paper describes a method for the selective precipitation and purification of a monovalent protein (carbonic anhydrase is used as a demonstration) from cellular lysate using ammonium sulfate and oligovalent ligands. The oligovalent ligands induce the formation of protein-ligand aggregates, and at an appropriate concentration of dissolved ammonium sulfate, these complexes precipitate. The purification involves three steps: (i) the removal of high-molecular-weight impurities through the addition of ammonium sulfate to the crude cell lysate; (ii) the introduction of an oligovalent ligand and the selective precipitation of the target protein-ligand aggregates from solution; and (iii) the removal of the oligovalent ligand from the precipitate by dialysis to release the target protein. The increase of mass and volume of the proteins upon aggregate formation reduces their solubility, and results in the selective precipitation of these aggregates. We recovered human carbonic anhydrase, from crude cellular lysate, in 82% yield and 95% purity with a trivalent benzene sulfonamide ligand. This method provides a chromatography-free strategy of purifying monovalent proteins--for which appropriate oligovalent ligands can be synthesized--and combines the selectivity of affinity-based purification with the convenience of salt-induced precipitation.     

Plasma glycoprotein profiling for colorectal cancer biomarker identification by lectin glycoarray and lectin blot

Colorectal cancer (CRC) remains a major worldwide cause of cancer-related morbidity and mortality largely due to the insidious onset of the disease. The current clinical procedures utilized for disease diagnosis are invasive, unpleasant, and inconvenient; hence, the need for simple blood tests that could be used for the early detection of CRC. In this work, we have developed methods for glycoproteomics analysis to identify plasma markers with utility to assist in the detection of colorectal cancer (CRC). Following immunodepletion of the most abundant plasma proteins, the plasma N -linked glycoproteins were enriched using lectin affinity chromatography and subsequently further separated by nonporous silica reversed-phase (NPS-RP)-HPLC. Individual RP-HPLC fractions were printed on nitrocellulose coated slides which were then probed with lectins to determine glycan patterns in plasma samples from 9 normal, 5 adenoma, and 6 colorectal cancer patients. Statistical tools, including principal component analysis, hierarchical clustering, and Z-statistics analysis, were employed to identify distinctive glycosylation patterns. Patients diagnosed with colorectal cancer or adenomas were shown to have dramatically higher levels of sialylation and fucosylation as compared to normal controls. Plasma glycoproteins with aberrant glycosylation were identified by nano-LC-MS/MS, while a lectin blotting methodology was used to validate proteins with significantly altered glycosylation as a function of cancer progression. The potential markers identified in this study for diagnosis to distinguish colorectal cancer from adenoma and normal include elevated sialylation and fucosylation in complement C3, histidine-rich glycoprotein, and kininogen-1. These potential markers of colorectal cancer were subsequently validated by lectin blotting in an independent set of plasma samples obtained from 10 CRC patients, 10 patients with adenomas, and 10 normal subjects. These results demonstrate the utility of this strategy for the identification of N -linked glycan patterns as potential markers of CRC in human plasma, and may have the utility to distinguish different disease states.     

Ants impact the composition of the aquatic macroinvertebrate communities of a myrmecophytic tank bromeliad

In an inundated Mexican forest, 89 out of 92 myrmecophytic tank bromeliads (Aechmea bracteata) housed an associated ant colony: 13 sheltered Azteca serica, 43 Dolichoderus bispinosus, and 33 Neoponera villosa. Ant presence has a positive impact on the diversity of the aquatic macroinvertebrate communities (n = 30 bromeliads studied). A Principal Component Analysis (PCA) showed that the presence and the species of ant are not correlated to bromeliad size, quantity of water, number of wells, filtered organic matter or incident radiation. The PCA and a generalized linear model showed that the presence of Azteca serica differed from the presence of the other two ant species or no ants in its effects on the aquatic invertebrate community (more predators). Therefore, both ant presence and species of ant affect the composition of the aquatic macroinvertebrate communities in the tanks of A. bracteata, likely due to ant deposition of feces and other waste in these tanks.