The Uba2 and Ufd1 proteins of Saccharomyces cerevisiae interact with poly(A) polymerase and affect the polyadenylation activity of cell extracts

Poly(A) polymerase is responsible for the addition of the adenylate tail to the 3′ ends of mRNA. Using the two-hybrid system, we have identified two proteins which interact specifically with the Saccharomyces cerevisiae poly(A) polymerase, Pap1. Uba2 is a homolog of ubiquitin-activating (E1) enzymes and Ufd1 is a protein whose function is probably also linked to the ubiquitin-mediated protein degradation pathway. These two proteins interact with Pap1 and with each other, but not with eight other target proteins which were tested in the two-hybrid system. The last 115 amino acids of Uba2, which contains an 82-amino acid region not present in previously characterized E1 enzymes, is sufficient for the interaction with Pap1. Both Uba2 and Ufd1 can be co-immunoprecipitated from extracts with Pap1, confirming in vitro the interaction identified by two-hybrid analysis. Depletion of Uba2 from cells produces extracts which polyadenylate precursor RNA with increased efficiency compared to extracts from nondepleted cells, while depletion of Ufd1 yields extracts which are defective in processing. These two proteins are not components of polyadenylation factors, and instead may have a role in regulating poly(A) polymerase activity. Received: 6 January 1997 / Accepted: 27 February 1997     

Fibroblastlike primary cells from human colon adenocarcinoma explants: Collagen biosynthesis

Summary Fibroblastlike primary cells have been obtained from human colon adenocarcinoma explants. Such cells disappear during cell culture and thus have not been previously studied. These cells have a number of altered phenotypic characteristics: a) morphology; b) growth behavior and adherence to culture substrate (they required 3 h for 90% attachment and only presented a flattened morphology 40 h after platting); and c) collagen metabolism. Increased protein biosynthesis (about double than control colon-derived fibroblasts) and maintained ability for collagen biosynthesis have been observed for the tumor-associated fibroblastlike cells. Thus, the collagen to noncollagenous proteins ratio was decreased for these cells. They exhibited an altered type I:type III collagen (5:1 instead of 3:1 in colon fibroblasts) and procollagen (2:1 against 5:1 in colon fibroblasts) ratios as well as a decreased secretion of collagen with an abnormal deposition of procollagens in the cell layer. These studies show a permanent phenotypic alteration in the tumor-associated fibroblastlike cells.     

Set Configuration in Resistance Exercise: Muscle Fatigue and Cardiovascular Effects

Purpose

Cardiovascular responses of traditional resistance (TS) training have been extensively explored. However, the fatigue mechanisms associated with an intra-set rest configuration (ISR) have not been investigated. This study compares two modalities of set configurations for resistance exercise that equates work to rest ratios and measures the central and peripheral fatigue in combination with cortical, hemodynamic and cardiovascular measures.

Methods

11 subjects performed two isometric knee extension training sessions using TS and ISR configurations. Voluntary activation (VA), single twitch amplitude, low frequency fatigue (LFF), Mwave, motor evoked potential (MEP), short intracortical inhibition (SICI), intracortical facilitation (ICF) and heart rate variability were evaluated before and after each training session. During each session beat to beat heart rate, blood pressure and rate pressure product (RPP) were also evaluated.

Results

After exercise VA decreased significantly for TS but not for ISR (P < 0.001), single twitch amplitude and LFF values were lower for TS than ISR (P < 0.004), and SICI was reduced only for the TS configuration (P = 0.049). During exercise RPP values were significantly higher for the TS than for ISR (P = 0.001). RPP correlated with VA for TS (r = -.85 P < 0.001) suggesting a relationship between central fatigue and cardiovascular stress.

Conclusions

We conclude that ISR induced lower central and peripheral fatigue as well as lower cardiovascular stress in comparison with TS configuration. Our study suggests that set configuration is a key factor in the regulation of the neuromuscular and cardiovascular responses of resistance training.     

Synthesis and antifungal activity of diverse C-2 pyridinyl and pyridinylvinyl substituted quinolines

Diverse 2-pyridinyl quinolines 6–12 and 2-pyridinilvinyl quinolines 13–17 were prepared using a straightforward synthesis based on the BiCl₃-catalyzed multicomponent imino Diels–Alder (imino DA) reaction or a novel tandem imino DA/catalytic tetrahydroquinoline ring oxidation/Perkin condensation sequential process. All members of the series showed activities against dermatophytes and some of them possessed a broad spectrum of action. 2-(Pyridin-4-yl)quinoline 9 and 2-(2-pyridin-4-yl)vinyl)quinoline 16 showed the best MIC₈₀ and MIC₅₀ against the clinically important fungi Candida albicans and non-albicans Candida species. In turn, 6-ethyl-2-(pyridin-2-yl)quinoline 6 showed the best properties against standardized as well as clinical strains of Cryptococcus neoformans.     

Short-term effects of litter from 21 woody species on plant growth and root development

Background and aims

Plant litter has an important role in terrestrial ecosystems (Lambers et al. 2008). Our aim was to assess the short-term effect of litter from 21 woody species (deciduous and evergreens) on plant growth and root development.

Methods

We conducted a short-term experiment (10 weeks) under controlled conditions adding litter from 21 woody species to pots with Dactylis glomerata (target species). We determined plant biomass and root development and related these variables to decomposition rate and litter quality.

Results

Litter from two species enhanced plant growth whereas litter of five species inhibited it. Considering all species in the data set, plant growth was associated to litter with high decomposition rate and high litter quality: high Ca and N concentration and low polyphenols concentration. However, excluding from the analyses the two species that increased growth, litter inhibition effect on plant growth was related to the litter-polyphenols concentration. Plants growing with nutrient-richer litter had a lower proportion of fine roots which could be related to a litter mediated increase in soil nutrient.

Conclusions

Enhanced plant growth or, on the contrary, plant growth inhibition could be the result of a positive or, in turn, negative balance between nutrient and polyphenols concentration in litter.     

Cellular characterisation of Candida tropicalis presenting fluconazole-related trailing growth

We assessed fluconazole susceptibility in 52 Candida tropicalis clinical strains using seven antifungal susceptibility methods, including broth microdilution (BMD) [standard M27 A3 (with neutral and acid pH), ATB Fungus 3, Vitek 2 system and flow cytometric analysis] and agar-based methods (disk diffusion and E-test). Trailing growth, detection of cell-associated secreted aspartic proteases (Saps) and morphological and ultrastructural traits of these clinical strains were also examined. The ranges of fluconazole 24 h-minimum inhibitory concentration (MIC) values were similar among all methods. The essential agreement among the methods used for MIC determinations was excellent and all methods categorised all strains as susceptible, except for one strain that showed a minor error. The presence of the trailing effect was assessed by six methods. Trailing positivity was observed for 86.5-100% of the strains. The exception was the BMD-Ac method where trailing growth was not observed. Morphological and ultrastructural alterations were detected in C. tropicalis trailing cells, including mitochondrial swelling and cell walls with irregular shapes. We tested the production of Saps in 13 C. tropicalis strains expressing trailing growth through flow cytometry. Our results showed that all of the C. tropicalis strains up-regulated surface Sap expression after 24 h or 48 h of exposure to fluconazole, which was not observed in untreated yeast strains. We concluded that C. tropicalis strains expressing trailing growth presented some particular features on both biological and ultrastructural levels.     

Non-surgical deep intrauterine transfer of superfine open pulled straw (SOPS)-vitrified porcine embryos: Evaluation of critical steps of the procedure

Previous trials achieved extremely poor results when using the one-step warming method in a syringe in combination with non-surgical deep intrauterine transfer (NET) of superfine open pulled straw (SOPS)-vitrified embryos. This study aimed to assess the effect of the warming procedure on the in vitro and in vivo development of SOPS-vitrified embryos. The effect of the passage of the vitrified-warmed (VW) embryos through the NET catheter was also evaluated. Groups of 4 to 6 morulae and blastocysts, collected from weaned sows, were SOPS-vitrified in 1 μL of vitrification medium, warmed by the one-step warming method in a dish or in a 1-mL syringe and cultured in vitro for 48 h to evaluate the embryo survival (ES) and hatching rates (HR). Warming in syringe had a deleterious effect (P < 0.05) on the in vitro ES (60.5 ± 10.4%) and HR (39.6 ± 9.5%) of VW embryos in comparison with embryos warmed in a dish (85.4 ± 10.6% and 69.0 ± 8.4%, respectively). This decreased embryonic development was due to the increased time required between the removal of the straws from the liquid nitrogen and the contact of the embryos with the warming medium when the warming was performed in a syringe in comparison with that for the warming in a dish. After verifying that the passage of VW embryos through the NET catheter does not have a damaging effect on their further in vitro development, the negative effect of warming in a syringe was also confirmed after NET. Fifteen fresh and SOPS-vitrified embryos warmed in a syringe or in a dish were transferred to each recipient (n = 28) and recovered 24 h later to assess their developmental progression. All embryos from the syringe group were found to have degenerated at recovery. The in vivo ES and HR from the dish group (80.4 ± 3.4% and 14.2 ± 7.2%, respectively) were lower (P < 0.05) than those from the fresh group (94.0 ± 4.1% and 36.8 ± 7.8%, respectively). Combining the warming in a dish and the NET procedure, 35 VW embryos were transferred to each of 10 gilts. Five recipients farrowed an average of 10.4 ± 0.9 piglets. In conclusion, the method of one-step warming in a syringe has a negative effect on the in vitro and in vivo viability of SOPS-vitrified porcine embryos. In addition, NET of SOPS-vitrified embryos warmed by the one-step method in a dish showed promising reproductive performance of recipients. However, despite the great potential of this technology, further developments are required for large-scale commercial applications.