Accumulation of uridine diphosphoglucose pyrophosphorylase in Dictyostelium discoideum via preferential synthesis

At the end of the exponential growth phase, the enzyme UDP-glueose pyrophosphorylase is present in the vegetative cells of Dictyostelium discoideum NC4 (haploid) at a low level (about 0.05% of total protein). During the initial stages of fruiting body construction, while the cells are entering into multicellular aggregates, the enzyme level remains constant, but increases dramatically thereafter reaching a peak (about 0.5% of total protein) at the end of fruiting body construction, and then partially decreasing. Previous studies have shown that both the accumulation and disappearance are keyed to the flow of morphogenetic events.In this study, cells were labeled with amino acids for different periods throughout the sequence. The enzyme was quantitatively immune-precipitated from crude cell extracts, the precipitate was washed and redissolved, and the enzyme protein separated by acrylamide gel electrophoresis in order to estimate the differential incorporation ratio, i.e. $\text{disints}\text{min in enzyme protein per 10}{}^8\text{cells}\text{disints}\text{min in total protein per 10}{}^8\text{cells}\text{× 100}$ for each labeling period. During the initial stages, when the enzyme level remained relatively constant, this ratio was about 0.03 to 0.04%. As the enzyme began to accumulate it rose progressively, attaining levels of 0.6 to 0.8% toward the end of fruiting body construction before declining. The data are not consistent with the theory of Gustafson and Wright (1973) that differential turnover controls the level of this enzyme during the development of D. discoideum. They are consistent with the conclusion that directed changes in the differential rate of synthesis of UDP-glucose pyrophosphorylase is the controlling element.The estimates of enzyme content are based on a value for the specific enzyme activity of 100,000 units/mg enzyme, which had been determined previously using samples of the enzyme purified to apparent physical homogeneity. This figure has been confirmed in the present study by quantitative immuneprecipitation of the enzyme from crude extracts of homogeneously labeled cells. The method can be generally used to determine if a specific biological activity estimate obtained with a purified protein is consistent with its activity when measured before or during purification.     

Cortisol stress response and histopathological alteration index in Clarias gariepinus exposed to sublethal concentrations of Qua Iboe crude oil and rig wash

The histological effect on and stress response of post juvenile Clarias gariepinus exposed to Qua Iboe crude oil and rig wash were investigated. Fish weighing 60–90 g and measuring 16–18 cm were exposed for 7–28 days to 8.00 ml^?1 Qua Iboe crude oil and 0.0018 ml^–1 rig wash, both being 0.1 of the 96 hr LC₅₀. Blood samples of C. gariepinus were collected every seven days and evaluated for stress by measuring cortisol concentration. The gills and liver were studied and scored for Gill Alteration Index (GAI) and Hepatic Alteration Index (HAI), respectively. There was an increase in cortisol level up to the 7th and 14th day among the group exposed to Qua Iboe crude oil, with a decrease on the 21st and 28th day. The rig wash group increased in cortisol level up to the 7th day and decreased slightly on the 14th day, after which the trend became irregular. The toxic effects of the Qua Iboe crude oil and rig wash were time dependent, as shown by the histopathological alteration index (HAI) of gill and liver. After 28 days of exposure, the gills had irreparable damage due to high frequency of cellular necrosis and degeneration, whereas the liver had from moderate to severe damage due to the high frequency of cellular degeneration and inflammation. Qua Iboe crude oil and rig wash are both toxic to C. gariepinus, therefore their indiscriminate discharge to the environment must be discouraged.     

Murine Dishevelled 3 Functions in Redundant Pathways with Dishevelled 1 and 2 in Normal Cardiac Outflow Tract,Cochlea, and Neural Tube Development

Dishevelled (Dvl) proteins are important signaling components of both the canonical β-catenin/Wnt pathway, which controls cell proliferation and patterning, and the planar cell polarity (PCP) pathway, which coordinates cell polarity within a sheet of cells and also directs convergent extension cell (CE) movements that produce narrowing and elongation of the tissue. Three mammalian Dvl genes have been identified and the developmental roles of Dvl1 and Dvl2 were previously determined. Here, we identify the functions of Dvl3 in development and provide evidence of functional redundancy among the three murine Dvls. Dvl3 ^−/− mice died perinatally with cardiac outflow tract abnormalities, including double outlet right ventricle and persistent truncus arteriosis. These mutants also displayed a misorientated stereocilia in the organ of Corti, a phenotype that was enhanced with the additional loss of a single allele of the PCP component Vangl2/Ltap (LtapLp/+). Although neurulation appeared normal in both Dvl3 ^−/− and LtapLp/+ mutants, Dvl3 ^+/−;LtapLp/+ combined mutants displayed incomplete neural tube closure. Importantly, we show that many of the roles of Dvl3 are also shared by Dvl1 and Dvl2. More severe phenotypes were observed in Dvl3 mutants with the deficiency of another Dvl, and increasing Dvl dosage genetically with Dvl transgenes demonstrated the ability of Dvls to compensate for each other to enable normal development. Interestingly, global canonical Wnt signaling appeared largely unaffected in the double Dvl mutants, suggesting that low Dvl levels are sufficient for functional canonical Wnt signals. In summary, we demonstrate that Dvl3 is required for cardiac outflow tract development and describe its importance in the PCP pathway during neurulation and cochlea development. Finally, we establish several developmental processes in which the three Dvls are functionally redundant.     

Use of a Bacteriophage Lysin,PlyC, as an Enzyme Disinfectant against Streptococcus equi

Streptococcus equi is the causative agent of the purulent infection equine strangles. This disease is transmitted through shedding of live bacteria from nasal secretions and abscess drainage or by contact with surfaces contaminated by the bacteria. Disinfectants are effective against S. equi, but inactivation by environmental factors, damage to equipment, and toxicity are of great concern. Bacteriophage-encoded lysins (cell wall hydrolases) have been investigated as therapeutic agents due to their ability to lyse susceptible gram-positive organisms. Here, we investigate the use of one lysin, PlyC, as a narrow-spectrum disinfectant against S. equi. This enzyme was active against >20 clinical isolates of S. equi, including both S. equi subsp. equi and S. equi subsp. zooepidemicus. Significantly, PlyC was 1,000 times more active on a per weight basis than Virkon-S, a common disinfecting agent, with 1 μg of enzyme able to sterilize a 10⁸ CFU/ml culture of S. equi in 30 min. PlyC was subjected to a standard battery of tests including the Use Dilution Method for Testing Disinfectants and the Germicidal Spray Products Test. Results indicate that aerosolized PlyC can eradicate or significantly reduce the S. equi load on a variety of materials found on common stable and horse-related equipment. Additionally, PlyC was shown to retain full activity under conditions that mimic a horse stable, i.e., in the presence of nonionic detergents, hard water, or organic materials. We propose PlyC as the first protein-based, narrow-spectrum disinfectant against S. equi, which may augment or supplement the use of broad-spectrum disinfectants in barns and stables where equine strangles is prevalent.Equine strangles is a highly contagious lymphadenitis of the head and neck that is uniquely caused by Streptococcus equi, predominantly S. equi subsp. equi, with some disease associated with S. equi subsp. zooepidemicus (24, 29). Progression of the purulent infection leads to acute swelling and subsequent abscess formation of the submaxillary, submandibular, and retropharyngeal lymph nodes causing a “strangling” of the pharynx, with severe cases necessitating a tracheotomy (24). Serious complications occur in approximately 20% of infected horses, and the overall mortality rate has been reported to be as high as 8% on farms where the infection is endemic (25). Even after recovery from infection, long-lasting, immune-mediated complications such as progressive muscle atrophy have been reported, which can adversely affect the career of a racing, show, or work horse (27).The route of S. equi transmission is through nasal secretions and drainage from abscesses. Infected horses can nasally shed bacteria for weeks, contaminating surfaces through which other horses can become infected. S. equi extract and attenuated-live vaccines exist, but they are often associated with abscess formation at the site of injection, short duration of immunity, poor efficacy, and the very real threat of a nascent infection from the vaccine (15, 24, 29). Thus, strangles continues to be a serious and widespread infectious disease of horses despite the presence of multiple commercially available vaccines.Strangles prevention strategies include good disinfection/hygiene practices, isolation of infected animals, and removal of equipment for sanitization where possible (9, 26; also R. E. Holland, D. G. Harris, and A. Monge, presented at the 52nd Annual Convention of the American Association of Equine Practitioners, San Antonio, TX, 2 to 6 December 2006). Current broad-spectrum disinfectants belong to one of several chemical categories including alcohols, aldehydes, biguanides, halogens, oxidizing agents, phenols, or quaternary ammonium compounds (6, 8). To various degrees, these compounds have been shown to be flammable, light sensitive, carcinogenic, corrosive to metals, irritating to mucous membranes, and/or toxic to livestock and humans (8, 10). Additionally, many factors that are often associated with cleaning stalls/barns (e.g., hard water, organic load, or detergents) can reduce or even ablate efficacy of chemical disinfectants (8). Importantly, studies have shown that these commonly used disinfectants can select for mutant bacteria with decreased susceptibility to biocides and antibiotics without compromising virulence (21).Recently, bacteriophage-encoded peptidoglycan hydrolases, collectively termed lysins and often referred to as “enzybiotics,” have been investigated as potential therapeutic agents against pathogens due to their ability to lyse the bacterial cell wall (12). These enzymes not only exert their lethal effects in the absence of bacteriophage (cause “lysis from without”) but also display specificity for a bacterial host, often for a particular genus, species, or even a subspecies depending on the lysin (11). For example, one lysin, PlyC, is known to lyse streptococcal species bearing a polyrhamnose epitope, which include group C streptococcus (i.e., S. equi subsp. equi) among other streptococci (19). As an adjunct to broad-spectrum disinfectants, we investigate here the use of the PlyC enzyme to help control the acquisition and spread of S. equi subsp. equi in horse stalls and barns.     

Individual- and Neighborhood-Level Predictors of Mortality in Florida Colorectal Cancer Patients

Purpose

We examined individual-level and neighborhood-level predictors of mortality in CRC patients diagnosed in Florida to identify high-risk groups for targeted interventions.

Methods

Demographic and clinical data from the Florida Cancer Data System registry (2007–2011) were linked with Agency for Health Care Administration and US Census data (n = 47,872). Cox hazard regression models were fitted with candidate predictors of CRC survival and stratified by age group (18–49, 50–64, 65+).

Results

Stratified by age group, higher mortality risk per comorbidity was found among youngest (21%), followed by middle (19%), and then oldest (14%) age groups. The two younger age groups had higher mortality risk with proximal compared to those with distal cancer. Compared with private insurance, those in the middle age group were at higher death risk if not insured (HR = 1.35), or received healthcare through Medicare (HR = 1.44), Medicaid (HR = 1.53), or the Veteran’s Administration (HR = 1.26). Only Medicaid in the youngest (52% higher risk) and those not insured in the oldest group (24% lower risk) were significantly different from their privately insured counterparts. Among 18–49 and 50–64 age groups there was a higher mortality risk among the lowest SES (1.17- and 1.23-fold higher in the middle age and 1.12- and 1.17-fold higher in the older age group, respectively) compared to highest SES. Married patients were significantly better off than divorced/separated (HR = 1.22), single (HR = 1.29), or widowed (HR = 1.19) patients.

Conclusion

Factors associated with increased risk for mortality among individuals with CRC included being older, uninsured, unmarried, more comorbidities, living in lower SES neighborhoods, and diagnosed at later disease stage. Higher risk among younger patients was attributed to proximal cancer site, Medicaid, and distant disease; however, lower SES and being unmarried were not risk factors in this age group. Targeted interventions to improve survivorship and greater social support while considering age classification may assist these high-risk groups.     

SAUR Inhibition of PP2C-D Phosphatases Activates Plasma Membrane H+-ATPases to Promote Cell Expansion in Arabidopsis

The plant hormone auxin promotes cell expansion. Forty years ago, the acid growth theory was proposed, whereby auxin promotes proton efflux to acidify the apoplast and facilitate the uptake of solutes and water to drive plant cell expansion. However, the underlying molecular and genetic bases of this process remain unclear. We have previously shown that the SAUR19-24 subfamily of auxin-induced SMALL AUXIN UP-RNA (SAUR) genes promotes cell expansion. Here, we demonstrate that SAUR proteins provide a mechanistic link between auxin and plasma membrane H⁺-ATPases (PM H⁺-ATPases) in Arabidopsis thaliana. Plants overexpressing stabilized SAUR19 fusion proteins exhibit increased PM H⁺-ATPase activity, and the increased growth phenotypes conferred by SAUR19 overexpression are dependent upon normal PM H⁺-ATPase function. We find that SAUR19 stimulates PM H⁺-ATPase activity by promoting phosphorylation of the C-terminal autoinhibitory domain. Additionally, we identify a regulatory mechanism by which SAUR19 modulates PM H⁺-ATPase phosphorylation status. SAUR19 as well as additional SAUR proteins interact with the PP2C-D subfamily of type 2C protein phosphatases. We demonstrate that these phosphatases are inhibited upon SAUR binding, act antagonistically to SAURs in vivo, can physically interact with PM H⁺-ATPases, and negatively regulate PM H⁺-ATPase activity. Our findings provide a molecular framework for elucidating auxin-mediated control of plant cell expansion.     

Differential Regulation of Cellular Senescence and Differentiation by Prolyl Isomerase Pin1 in Cardiac Progenitor Cells

Autologous c-kit⁺ cardiac progenitor cells (CPCs) are currently used in the clinic to treat heart disease. CPC-based regeneration may be further augmented by better understanding molecular mechanisms of endogenous cardiac repair and enhancement of pro-survival signaling pathways that antagonize senescence while also increasing differentiation. The prolyl isomerase Pin1 regulates multiple signaling cascades by modulating protein folding and thereby activity and stability of phosphoproteins. In this study, we examine the heretofore unexplored role of Pin1 in CPCs. Pin1 is expressed in CPCs in vitro and in vivo and is associated with increased proliferation. Pin1 is required for cell cycle progression and loss of Pin1 causes cell cycle arrest in the G₁ phase in CPCs, concomitantly associated with decreased expression of Cyclins D and B and increased expression of cell cycle inhibitors p53 and retinoblastoma (Rb). Pin1 deletion increases cellular senescence but not differentiation or cell death of CPCs. Pin1 is required for endogenous CPC response as Pin1 knock-out mice have a reduced number of proliferating CPCs after ischemic challenge. Pin1 overexpression also impairs proliferation and causes G₂/M phase cell cycle arrest with concurrent down-regulation of Cyclin B, p53, and Rb. Additionally, Pin1 overexpression inhibits replicative senescence, increases differentiation, and inhibits cell death of CPCs, indicating that cell cycle arrest caused by Pin1 overexpression is a consequence of differentiation and not senescence or cell death. In conclusion, Pin1 has pleiotropic roles in CPCs and may be a molecular target to promote survival, enhance repair, improve differentiation, and antagonize senescence.