Gz, a guanine nucleotide-binding protein with unique biochemical properties

Cloning of a complementary DNA (cDNA) for Gz alpha, a newly appreciated member of the family of guanine nucleotide-binding regulatory proteins (G proteins), has allowed preparation of specific antisera to identify the protein in tissues and to assay it during purification from bovine brain. Additionally, expression of the cDNA in Escherichia coli has resulted in the production and purification of the recombinant protein. Purification of Gz from bovine brain is tedious, and only small quantities of protein have been obtained. The protein copurifies with the beta gamma subunit complex common to other G proteins; another 26- kDa GTP-binding protein is also present in these preparations. The purified protein could not serve as a substrate for NAD-dependent ADP- ribosylation catalyzed by either pertussis toxin or cholera toxin. Purification of recombinant Gz alpha (rGz alpha) from E. coli is simple, and quantities of homogeneous protein sufficient for biochemical analysis are obtained. Purified rGz alpha has several properties that distinguish it from other G protein alpha subunit polypeptides. These include a very slow rate of guanine nucleotide exchange (k = 0.02 min^-1), which is reduced greater than 20-fold in the presence of mM concentrations of Mg2+. In addition, the rate of the intrinsic GTPase activity of Gz alpha is extremely slow. The hydrolysis rate (kcat) for rGz alpha at 30 degrees C is 0.05 min^-1, or 200-fold slower than that determined for other G protein alpha subunits. rGz alpha can interact with bovine brain beta gamma but does not serve as a substrate for ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. These studies suggest that Gz may play a role in signal transduction pathways that are mechanistically distinct from those controlled by the other members of the G protein family

Analysis of the kinetic mechanism of recombinant human isoprenylcysteine carboxylmethyltransferase (Icmt)

BACKGROUND: Isoprenylcysteine carboxyl methyltransferase (Icmt) is the third of three enzymes that posttranslationally modify proteins that contain C-terminal CaaX motifs. The processing of CaaX proteins through this so-called prenylation pathway via a route initiated by addition of an isoprenoid lipid is required for both membrane targeting and function of the proteins. The involvement of many CaaX proteins such as Ras GTPases in oncogenesis and other aberrant proliferative disorders has led to the targeting of the enzymes involved in their processing for therapeutic development, necessitating a detailed understanding of the mechanisms of the enzymes. RESULTS: In this study, we have investigated the kinetic mechanism of recombinant human Icmt. In the reaction catalyzed by Icmt, S-adenosyl-L-methionine (AdoMet) provides the methyl group that is transferred to the second substrate, the C-terminal isoprenylated cysteine residue of a CaaX protein, thereby generating a C-terminal prenylcysteine methyl ester on the protein. To facilitate the kinetic analysis of Icmt, we synthesized a new small molecule substrate of the enzyme, biotin-S-farnesyl-L-cysteine (BFC). Initial kinetic analysis of Icmt suggested a sequential mechanism for the enzyme that was further analyzed using a dead end competitive inhibitor, S-farnesylthioacetic acid (FTA). Inhibition by FTA was competitive with respect to BFC and uncompetitive with respect to AdoMet, indicating an ordered mechanism with SAM binding first. To investigate the order of product dissociation, product inhibition studies were undertaken with S-adenosyl-L-homocysteine (AdoHcy) and the N-acetyl-S-farnesyl-L-cysteine methylester (AFCME). This analysis indicated that AdoHcy is a competitive inhibitor with respect to AdoMet, while AFCME shows a noncompetitive inhibition with respect to BFC and a mixed-type inhibition with respect to AdoMet. These studies established that AdoHcy is the final product released, and that BFC and AFCME bind to different forms of the enzyme. CONCLUSIONS: These studies establish that catalysis by human Icmt proceeds through an ordered sequential mechanism and provide a kinetic framework for analysis of specific inhibitors of this key enzyme

Habitat Use by Juvenile Gag, Mycteroperca microlepis (Pisces: Serranidae), in Subtropical Charlotte Harbor, Florida (USA)

Estuaries play a key role in the juvenile stage of gag (Myeteroperca microlepis). The use of estuarine habitats by juvenlle gag has been examined in temperate estuaries, which are at the northern limits of the range of this species, but the importance of subtropical estuaries during the early life history of this species has not been studied extensively. Gag were collected in subtropical Charlotte Harbor, Florida, during routine monthiy sampling from January 1996 to Decemher 2002. Juvenlle gag were collected using a 21.3-m seine, a 183-m haul seine, and a 183-m purse seine. A total of 738 individuals ranging from 30 to 489 mm standard length (SL) were collected in 4,480 samples. Most gag (96%) were probably young-of-the-year (\u3c 288 mm SL). The majority of juveniles were collected in polyhaline Gasparilla and Pine Island sounds from April to December, with a few larger individuals captured year-round. The observed period of gag settlement was similar to thet reported in other subtropical and temperate estuaries, but gag in Charlotte Harbor remained in the estuary longer and egressed at a larger size than did gag in other estuaries. Relative abundance of juvenile gag within Charlotte Harbor was greatest on shallow seagrass shoals but was also high along flinging mangrove shorelines, which is a habitat not previously reported for gag

Cold Atmospheric Plasma Induces Silver Nanoparticle Uptake, Oxidative Dissolution and Enhanced Cytotoxicity in Glioblastoma Multiforme Cells

Silver nanoparticles (AgNP) emerged as a promising reagent for cancer therapy with oxidative stress implicated in the toxicity. Meanwhile, studies reported cold atmospheric plasma (CAP) generation of reactive oxygen and nitrogen species has selectivity towards cancer cells. Gold nanoparticles display synergistic cytotoxicity when combined with CAP against cancer cells but there is a paucity of information using AgNP, prompting to investigate the combined effects of CAP using dielectric barrier discharge system (voltage of 75 kV, current is 62.5 mA, duty cycle of 7.5kVA and input frequency of 50–60Hz) and 10 nm PVA-coated AgNP using U373MG Glioblastoma Multiforme cells. Cytotoxicity in U373MG cells was \u3e100-fold greater when treated with both CAP and PVA-AgNP compared with either therapy alone (IC50 of 4.30 μg/mL with PVA-AgNP alone compared with 0.07 μg/mL after 25s CAP and 0.01 μg/mL 40s CAP). Combined cytotoxicity was ROS-dependent and was prevented using N-Acetyl Cysteine. A novel darkfield spectral imaging method investigated and quantified AgNP uptake in cells determining significantly enhanced uptake, aggregation and subcellular accumulation following CAP treatment, which was confirmed and quantified using atomic absorption spectroscopy. The results indicate that CAP decreases nanoparticle size, decreases surface charge distribution of AgNP and induces uptake, aggregation and enhanced cytotoxicity in vitro

Where does stress happen? Ecological momentary assessment of daily stressors using a mobile phone app. [Journal article]

Despite the importance of daily stress to individuals' health and wellbeing, few studies have explored where stress happens in real time, that is, dynamic stress processes in different spaces. As such, stress interventions rarely account for the environment in which stress occurs. We used mobile phone based ecological momentary assessment (EMA) to collect daily stress data. Thirty-three participants utilized a mobile-phone-based EMA app to self-report stressors as they went about their daily lives. Geographic coordinates were automatically collected with each stress report. Data from thematic analysis of stressors by location (home, work, work from home, other) were used to determine whether certain stressors were more prevalent in certain environments. Nine daily stressors significantly differed by location. Work-related stress was reported more often at work. Pets, household chores, sleep, and media-related stressors were reported most at home. Physical illnesses, vehicle issues, and safety/security stressors occurred most often while participants were "working from home." Traffic-related stress was experienced more commonly in "other" environments. Other 18 stressors were generated regardless of location, suggesting that these stressors were persistent and without respect to location. Study findings expand the understanding of environments in which specific stressors occur, providing baseline data for potential targeted "just-in-time" stress interventions tailored to unique stressors in specific environments. We also provide findings related to the "work from home" phenomenon. Further work is needed to better understand the unique stressors among the large number of individuals who transitioned to working from home during and after the COVID-19 pandemic

Analytical sensitivity of COVID-19 rapid antigen tests: A case for a robust reference standard

Aggressive diagnostic testing remains an indispensable strategy for health and aged care facilities to prevent the transmission of SARS-CoV-2 in vulnerable populations. The preferred diagnostic platform has shifted towards COVID-19 rapid antigen tests (RATs) to identify the most infectious individuals. As such, RATs are being manufactured faster than at any other time in our history yet lack the relevant quantitative analytics required to inform on absolute analytical sensitivity enabling manufacturers to maintain high batch-to-batch reproducibility, and end-users to accurately compare brands for decision making. Here, we describe a novel reference standard to measure and compare the analytical sensitivity of RATs using a recombinant GFP-tagged nucleocapsid protein (NP-GFP). Importantly, we show that the GFP tag does not interfere with NP detection and provides several advantages affording streamlined protein expression and purification in high yields as well as faster, cheaper and more sensitive quality control measures for post-production assessment of protein solubility and stability. Ten commercial COVID-19 RATs were evaluated and ranked using NP-GFP as a reference standard. Analytical sensitivity data of the selected devices as determined with NP-GFP did not correlate with those reported by the manufacturers using the median tissue culture infectious dose (TCID50) assay. Of note, TCID50 discordance has been previously reported. Taken together, our results highlight an urgent need for a reliable reference standard for evaluation and benchmarking of the analytical sensitivity of RAT devices. NP-GFP is a promising candidate as a reference standard that will ensure that RAT performance is accurately communicated to healthcare providers and the public

Enhanced Bovine Colostrum Supplementation Shortens the Duration of Respiratory Disease in Thoroughbred Yearlings

AbstractBovine colostrum (BC) is used in humans as a nutritional supplement for immune support and has been shown to reduce Respiratory disease (RD). Other nutritional supplements, minerals and vitamins including mannan oligosaccharides (MOS), zinc and vitamins A, C and E have also been used for immune support. The aim of this prospective blinded randomized clinical trial was to evaluate the effects of a BC, MOS, zinc and vitamin based enhanced bovine colostrum supplement (BCS) on incidence and duration of RD occurring in yearling horses. 109 yearlings on two Thoroughbred farms in Central Kentucky were randomly assigned to treatment or placebo groups. Yearlings were supplemented once daily for 17 to 25 weeks with 100 g of a high quality commercial BCS (containing 50 g BC) or a full fat soy flour placebo, which were applied as a “top-dress” to feed. Yearlings were observed daily and evaluated weekly for signs of RD. All yearlings completed the study. The proportion of the study period during which each yearling exhibited illness was considerably shorter for BCS yearlings (least squares mean = 23% of the study period) than placebo yearlings (least squares mean = 34% of the study period, P = .002). The average duration of illness was shorter for BCS yearlings (1.96 weeks) than placebo yearlings (4.39 weeks, P < .0001). There was no statistical difference in the incidence of RD in these study yearlings

Farnesylation of YDJ1p is required for function at elevated growth temperatures in Saccharomyces cerevisiae

The Saccharomyces cerevisiae YDJ1 protein (YDJ1p) contains a C-terminal "CaaX box" motif common to proteins that are modified by prenylation. In the present study we show that YDJ1p is a specific substrate for both yeast and mammalian protein farnesyltransferase enzymes in vitro. A mutant form of YDJ1p, in which the conserved cysteine of the CaaX box is mutated to a serine (ydj1-S406p), cannot be farnesylated in vitro. After expression in S. cerevisiae, ydj1-S406p displays a reduced electrophoretic mobility and an increased cytosolic localization in subcellular fractionation experiments when compared to wild type YDJ1p. Expression of ydj1-S406 in cells lacking YDJ1 results in a temperature-sensitive growth phenotype in S. cerevisiae. These data indicate that farnesylation of YDJ1p is required for its function at elevated temperature