Light induction of the clock-controlled geneccg-1 is not transduced through the circadian clock inNeurospora crassa

Ambient light and the circadian clock have been shown to be capable of acting either independently or in an interrelated fashion to regulate the expression of conidiation in the ascomycete fungusNeurospora crassa. Recently several molecular correlates of the circadian clock have been identified in the form of the morning-specific clock-controlled genesccg-1 andccg-2. In this paper we report studies on the regulation ofccg-1, an abundantly expressed gene displaying complex regulation. Consistent with an emerging consensus for clock-controlled genes and conidiation genes inNeurospora, we report thatccg-1 expression is induced by light, and show that this induction is independent of the direct effects of light on the circadian clock. Although circadian regulation of the gene is lost in strains lacking a functional clock, expression ofccg-1 is still not constitutive, but rather fluctuates in concert with changes in developmental potential seen in such strains. Light induction ofccg-1 requires the products of theNeurospora wc-1 andwc-2 genes, but surprisingly the requirement forwc-2 is suppressed in conditional mutants ofcot-1, a gene that encodes a cAMP-dependent protein kinase. These data provide insight into a complex regulatory web, involving at least circadian clock control, light control, metabolic control, and very probably developmental regulation, that governs the expression ofccg-1.     

A Simple Purification of Indole-3-Acetic Acid and Abscisic Acid for GC-SIM-MS Analysis by Microfiltration of Aqueous Samples through Nylon

A simple procedure was developed for the partial purification of plant tissue samples to be analyzed simultaneously for indole-3-acetic acid (IAA) and abscisic acid (ABA). The procedure relies on removal of contaminants by filtration through nylon and partitioning into dichloromethane. This procedure successfully purified both IAA and ABA from muskmelon, cotton, and broccoli tissue. Twenty individual samples can be purified and methylated in 8 h for analysis of free IAA and ABA with gas chromatography-selected ion monitoring-mass spectrometry. The use of microfiltration of aqueous samples through nylon offers new opportunities for improving the efficiency of existing sample purification procedures.     

Nongenomic inhibition of oxytocin binding by progesterone in the ovine uterus

Progesterone (P4) has been reported to inhibit oxytocin (OT) binding to its receptor in isolated murine endometrial membranes. The purpose of the present research was to 1). examine the in vivo and in vitro effect of P4 on the binding of OT to its receptor in the ovine endometrium and 2). determine whether the endometrial plasma membranes have high-affinity binding sites for P4. Ovariectomized ewes were pretreated with a sequence of estradiol-17beta (2 days) and P4 (5 days) before being treated with estradiol-17beta plus either vehicle (corn oil), P4, or P4 + mifepristone (RU 486) for 3 consecutive days. Treatment of ewes with 10 mg P4/day for 3 days suppressed binding of OT (P < 0.01) compared with that of controls, whereas concomitant treatment with the progestin antagonist RU 486 (10 mg/day) blocked the effect of P4. Similarly, incubation of endometrial plasma membranes with P4 (5 ng/ml) inhibited binding of OT (P < 0.05), whereas this effect of P4 was blocked by the presence of RU 486 (10 ng/ml). By radioreceptor assay, the endometrial plasma membranes were found to contain a high-affinity binding site for P4 and the progestin agonist promegestone (Kd 1.2 x 10-9 and 1.74 x 10-10M, respectively). Incubation of endometrial plasma membranes with P4 (5 ng/ml) significantly increased the concentration of progestin binding sites. Binding of labeled promegestone (R 5020) was competitively inhibited by excess unlabeled R 5020, P4, RU 486, and OT but not by estradiol-17beta, cortisol, testosterone, and arginine vasopressin. These data suggest a direct suppressive action of P4 on the binding of OT to OT receptors in the ovine endometrial plasma membrane.     

The consequence of peroxidase overexpression in transgenic plants on root growth and development

Transgenic tobacco plants that overproduce the tobacco anionic peroxidase wilt upon reaching maturity, although having functional stomata and normal vascular anatomy and physiology. These plants were examined further to determine the cause for wilting, and thus better understand how the anionic peroxidase functions in plant growth and development. Shoots from young peroxidase overproducing plants were grafted onto wild-type tobacco root stock to determine if the roots could absorb and transmit sufficient water to maintain leaf turgidity. These grafted plants never wilted when grown in the greenhouse though shoot peroxidase activity remained ten-fold greater than in control plants, thus indicating that wilting is a consequence of peroxidase expression in the roots. Close examination of root systems revealed considerably less root mass in the transformed plant, primarily exhibited through a decrease in branching. At flowering, root growth rate and total root mass in transformed plants were less than 50% of control plants although shoot mass and growth rate were unchanged. This is in contrast to root growth in young seedlings where transformed plants performed equivalently to controls. Root hydraulic conductivity was measured to evaluate the effect of elevated peroxidase expression on water absorption and transport; however, no significant change in hydraulic conductivity was found in transformed plants. The consequence of anionic peroxidase overexpression on indoleacetic acid (IAA) metabolism was also examined. No significant difference in IAA levels was observed; however, root elongation in plants overexpressing peroxidase was insensitive to exogenous IAA. It can be concluded that the overexpression of the tobacco anionic peroxidase in transformed plants results in diminished root mass from fewer root branches, which contributes to the wilting phenomenon seen in these plants. Further, this developmental change in transformed plants may be a consequence of the metabolism of IAA by the anionic peroxidase.     

Cytokine profile of autologous conditioned serum for treatment of osteoarthritis, <Emphasis Type="Italic">in vitro</Emphasis>effects on cartilage metabolism and intra-articular levels after injection

Introduction  

Intraarticular administration of autologous conditioned serum (ACS) recently demonstrated some clinical effectiveness in treatment of osteoarthritis (OA). The current study aims to evaluate the in vitro effects of ACS on cartilage proteoglycan (PG) metabolism, its composition and the effects on synovial fluid (SF) cytokine levels following intraarticular ACS administration.     

Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri.

Vibrio fischeri is found both as a free-living bacterium in seawater and as the specific, mutualistic light organ symbiont of several fish and squid species. To identify those characteristics of symbiosis-competent strains that are required for successful colonization of the nascent light organ of juvenile Euprymna scolopes squids, we generated a mutant pool by using the transposon Mu dI 1681 and screened this pool for strains that were no longer motile. Eighteen independently isolated nonmotile mutants that were either flagellated or nonflagellated were obtained. In contrast to the parent strain, none of these nonmotile mutants was able to colonize the juvenile squid light organ. The flagellated nonmotile mutant strain NM200 possessed a bundle of sheathed polar flagella indistinguishable from that of the wild-type strain, indicating that the presence of flagella alone is not sufficient for colonization and that it is motility itself that is required for successful light organ colonization. This study identifies motility as the first required symbiotic phenotype of V. fischeri.     

Total antioxidant power in sled dogs supplemented with blueberries and the comparison of blood parameters associated with exercise

Oxidative damage from free radicals plays an important role in several diseases such as cancer, Alzheimer's disease, and heart disease. Research indicates that exercise contributes to oxidative stress. Fruits, such as blueberries, are good antioxidants because they contain phenolics that preferentially react with free radicals. Maintaining antioxidant levels by supplementing the diet with blueberries may prevent exercise-induced oxidative damage. The goal of our study was to compare antioxidant levels in sled dogs supplemented with blueberries on blood parameters within 48 h post-exercise. Though the exercise protocol did not cause unusual muscle damage as reflected in plasma creatine kinase and isoprostane levels, blueberry supplementation did elicit significantly elevated antioxidant status in sled dogs post exercise. This suggests that dogs fed blueberries while exercising as compared to dogs fed a control diet while exercising, may be better protected against oxidative damage.     

Catalytic cysteine of thymidylate synthase is activated upon substrate binding

The role of Ser 167 of Escherichia coli thymidylate synthase (TS) in catalysis has been characterized by kinetic and crystallographic studies. Position 167 variants including S167A, S167N, S167D, S167C, S167G, S167L, S167T, and S167V were generated by site-directed mutagenesis. Only S167A, S167G, S167T, and S167C complemented the growth of thymidine auxotrophs of E. coli in medium lacking thymidine. Steady-state kinetic analysis revealed that mutant enzymes exhibited k(cat) values 1.1-95-fold lower than that of the wild-type enzyme. Relative to wild-type TS, K(m) values of the mutant enzymes for 2'-deoxyuridylate (dUMP) were 5-90 times higher, while K(m) values for 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were 1.5-16-fold higher. The rate of dehalogenation of 5-bromo-2'-deoxyuridine 5'-monophosphate (BrdUMP), a reaction catalyzed by TS that does not require CH(2)H(4)folate as cosubstrate, by mutant TSs was analyzed and showed that only S167A and S167G catalyzed the dehalogenation reaction and values of k(cat)/K(m) for the mutant enzymes were decreased by 10- and 3000-fold, respectively. Analysis of pre-steady-state kinetics of ternary complex formation revealed that the productive binding of CH(2)H(4)folate is weaker to mutant TSs than to the wild-type enzyme. Chemical transformation constants (k(chem)) for the mutant enzymes were lower by 1.1-6.0-fold relative to the wild-type enzyme. S167A, S167T, and S167C crystallized in the I2(1)3 space group and scattered X-rays to either 1.7 A (S167A and S167T) or 2.6 A (S167C). The high-resolution data sets were refined to a R(crys) of 19.9%. In the crystals some cysteine residues were derivatized with 2-mercaptoethanol to form S,S-(2-hydroxyethyl)thiocysteine. The pattern of derivatization indicates that in the absence of bound substrate the catalytic cysteine is not more reactive than other cysteines. It is proposed that the catalytic cysteine is activated by substrate binding by a proton-transfer mechanism in which the phosphate group of the nucleotide neutralizes the charge of Arg 126', facilitating the transfer of a proton from the catalytic cysteine to a His 207-Asp 205 diad via a system of ordered water molecules.     

ISOLATION OF A NON‐PHAGE‐LIKE LYTIC VIRUS INFECTING AUREOCOCCUS ANOPHAGEFFERENS1

We have been working to characterize viruses that infect the HAB‐forming pelagophyte Aureococcus anophagefferens Hargraves et Sieburth. Field samples were collected during brown‐tide events in 2002 and tested for the presence of lytic agents. Here, we describe a recently isolated, lytic virus‐like particle (VLP) that is morphologically similar to particles observed in thin sections of infected A. anophagefferens cells from natural samples. TEM and SEM have revealed VLPs consistent with the morphological characteristics of previously described Phycodnaviridae. Large icosahedral particles (～140 nm) of similar shape and morphology dominate cell lysates and are accompanied by smaller phage‐like particles and heterotrophic prokaryotes that appear to be incurable from our cultures. To determine which of these particles interacts with the Aureococcus cells, we preserved cultures during the early stage of infection so that SEM could be used to visualize those particles that attach to the surface of naïve cultures. SEM revealed that 63% of the large icosahedral‐shaped particles attached to A. anophagefferens cells after only 30 min of exposure, while no significant frequency of attachment to the alga was observed for the phage‐like particles. The results of these observations are in contrast to previous studies, where phage‐like particles were reported to infect cells. When considered in conjunction with field observations, the results suggest that this newly isolated virus represents the dominant virus‐morphotype associated with bloom collapse and termination.