bluuebunny/crossref_metadata_2025 huggingface dataset:
The immediate effects of abrupt diet composition changes in young pigs
Authors: Kyriazakis I. , Emmans G. C.
Published: May 2005

Forty pigs (twenty males and twenty females) were weaned at 4 weeks of age into individual cages, and their weights and feed intakes measured daily. From weaning to 16 kg live weight they were given free access to a feed with either 134 (L) or 278 (H) g crude protein (nitrogen × 6.25)/kg fresh weight. During this period pigs on feed L grew at a slower rate and converted feed less efficiently than pigs on feed H. At 16 kg live weight they were introduced to a 6 d period of feeding when, on succesive days, they were given feeds L, H, L, H, L and H (if previously fed on L) or feeds H, L, H, L, H and L (if previously fed on H). For pigs previously given access to feed L the live-weight gain was 1061 v. 575 g/d on the days when H or L was given; the feed intake was 1078 v. 1027 g/d respectively. For pigs previously given access to feed H, the live-weight gain was 655 v. 610 g/d and the feed intake 844 v. 1071 g/d on the days when H or L were given. The interactions between the feed given previously and the feed given for the 3 d of the subsequent 6 d period were highly significant for both live-weight and feed intake. These rapid changes in feed intake and growth rate suggest that the metabolism of young pigs is extremely flexible, with a rapid rate of response to a change in the protein content of their feed.
The effect of fishmeal supplementation of a straw-based diet on growth and calorimetric efficiency of growth in heifers
Authors: Ortigues Isabelle , Smith T. , Gill M. , Cammell S. B. , Yarrow N. W.
Published: May 2005

Thirty-two 160 kg dairy heifers were used to measure the effects of increasing dietary protein content on growth and heat production. A basal diet containing (g/kg) 550 sodium hydroxide-treated straw, 220 barley, 220 sugarbeet pulp and 10 urea was offered with 0, 76 and 152 g fishmeal/kg dry matter of the basal diet (F0, F1 and F2 levels respectively). The three diets were each given at two levels of feeding (low, L; high, H): 57.6 g/d per kg metabolic body-weight (W0.75) for the LF0 diet and 74.7 g/d per kg W0.75 for the HFO diet. Apparent digestibility of the diets increased in response to the addition of fishmeal. Mean dry matter digestibility values were 0.67, 0.67, 0.69, 0.66, 0.68 and 0.69 and those for acid-detergent fibre digestibility were 0.60, 0.63, 0.66, 0.58, 0.60 and 0.65 for diets LF0, LF1, LF2, HF0, HF1 and HF2 respectively. Nitrogen retention increased in response to both fishmeal and feeding level. Live-weight gains were 170, 296, 434 g/d for the LF0, LF1 and LF2 diets and 468, 651 and 710 g/d for the HF0, HF1 and HF2 diets respectively. There were significant effects of increasing the plane of feeding and the level of fishmeal in the diet on live-weight gain. Dietary effects on live-weight gains were accompanied by increases in mean energy retention of 23, 45, 82, 94, 160 and 152 kJ/d per kg W0.75 for diets LF0, LF1, LF2, HF0, HF1 and HF2 respectively, but no definite evidence was obtained that dietary supplementation with fishmeal modified the efficiency of utilization of metabolizable energy for growth.
Comparison of growth, nitrogen metabolism and organ weights in piglets and rats fed on diets containingPhaseolus vulgarisbeans
Authors: Huisman J. , Der Poel A. F. B. Van , Leeuwen P. Van , Verstegen M. W. A.
Published: May 2005

The effects of lectins in the diet have been mainly studied in rats. An important question is whether results obtained in rats can be extrapolated to larger animals like the pig.Phaseolus vulgarisbeans are rich in toxic lectins. Therefore a study was carried out to compare the effects of diets containing 200 gPhaseolus vulgarisbeans (raw or toasted)/kg in rats and piglets. Live-weight gain, nitrogen digestibility and N balance were much lower in piglets than in rats fed on diets containing raw beans. Live-weight gain and N balance were slightly negative in the piglets. When toasted beans were given, live-weight gain and N balance values were reduced in piglets but hardly at all in rats. Giving raw beans caused hypertrophy of the pancreas in the rats but in piglets the weight of the pancreas was reduced. Spleen weight was depressed in the piglets but not in the rats. Weight of liver was not affected in either animal species. When toasted beans were given no effects on the weights of pancreas, spleen or liver were found in piglets or rats. It was concluded that the piglet is much more sensitive to antinutritional factors in thePhaseolus vulgarisbean than the rat.
Effect of variable protein contents in diets containing Phaseolus vulgaris beans on performance, organ weights and blood variables in piglets, rats and chickens
Authors: Huisman J. , Der Poel A. F. B. Van , Mouwen J. M. V. M. , WEERDEN J. M. V. M.
Published: May 2005

A comparison was made of the effects of antinutritional factors present in Phaseolus vulgaris on piglets, rats and chickens. Also the hypothesis of whether the negative effect on weight gain due to the inclusion of raw Phaseolus vulgaris in the diet can be attributed to an insufficient supply of amino acids was tested. Test diets containing 200 g raw Phaseolus beans/kg were balanced for digestible protein and amino acids; in one diet extra casein was incorporated. The main response criteria were live-weight gain and the weight of various organs including the intestine. Live-weight gain in piglets was markedly reduced during feeding 200 g raw Phaseolus vulgaris/kg in the diet, but not in rats and chickens. Addition of casein did not improve the weight gain of the piglets, indicating that a toxic factor was responsible for the reduced weight gain and not an insufficient supply of amino acids. The weights of the spleen and thymus were markedly reduced in the piglets when the diets with raw Phaseolus beans were given, but not in the rats and chickens. Additional supply of casein did not change this effect. Indications were found that when the supply of dietary protein is adequate there is no reduction in pancreas weight with raw Phaseolus beans as was observed in previous experiments. The weight of the intestine was increased in all three species due to feeding raw Phaseolus vulgaris.
Effect of defaunation on protein and fibre digestion in sheep fed on ammonia-treated straw-based diets with or without maize
Authors: Ushida K. , Kayouli C. , De Smet S. , Jouany J. P.
Published: May 2005

Using a defaunating method which preserved bacteria and fungi in the rumen, the effect of protozoa on protein and fibre digestion was studied in six adult wethers in relation to the nature of the diet. Sheep were given daily, 42 g dry matter (DM)/kg metabolic body-weight (W0.75), one of two isonitrogenous diets: one contained ammonia-treated wheat straw as the only energy source (diet S) and the other was supplemented with maize grain pellets (diet SM). Mean daily intakes (g/d) of nitrogen, neutral-detergent fibre and acid-detergent fibre were respectively 22, 573 and 373 for diet S and 23, 450 and 334 for diet SM. Elimination of protozoa increased duodenal non-ammonia-nitrogen flow. This result was mainly due to an increase in microbial protein flow and, to a lesser extent, to a higher dietary protein flow. Defaunation markedly increased the efficiency of microbial protein synthesis. Maize-grain supplementation had a net positive effect on this variable in defaunated sheep, but not in faunated sheep. Cell-wall carbohydrates were less well digested in the defaunated rumen, and the negative effect of defaunation was greatest with the diet SM. Intestinal fibre digestion increased in the defaunated sheep especially in those fed on diet SM, but not enough to compensate for the decrease in rumen digestion.
Nutritional implications of D-xylose in pigs
Authors: Schutte J. B. , de Jong J. , Polziehn R. , Verstegen M. W. A.
Published: May 2005

Hemicellulose consists primarily of pentose sugars, joined together in a polysaccharide chain with d-xylose as the most abundant component. Ileal digestibility and urinary excretion of d-xylose and associated effects of this pentose sugar on ileal and faecal digestibility of dry matter (DM), organic matter (OM), gross energy (GE) and nitrogen were studied in pigs. Castrated pigs were prepared with a post-valvular T-caecum cannula to measure ileal digestibility. Faecal digestibility was measured in non-cannulated pigs. d-xylose was given at dietary inclusion levels of 100 and 200 g/kg, and the control sugar, d-glucose, at a rate of 200 g/kg diet. Ileal digestibility of d-xylose as well as that of d-glucose was found to be close to 100%. The presence of d-xylose in the diet decreased ileal digesta pH and increased ileal flow of volatile fatty acids, suggesting the occurrence of microbial degradation of d-xylose in the pig small intestine. In pigs fed on the 100 g d-xylose/kg diet, 44.5% of the d-xylose intake appeared in the urine. This percentage increased significantly to 52.6 when pigs were fed on the 200 g d-xylose/kg diet. Ileal and faecal digestibility of DM, OM, GE and N, as well as N retention, decreased significantly in pigs fed on the 200 g d-xylose/kg diet.
Nitrogen transactions along the digestive tract of lambs concurrently infected with Trichostrongylus colubriformis and Ostertagia circumcincta
Authors: Bown M. D. , Poppi D. P. , Sykes A. R.
Published: May 2005

Twelve lambs, paired on the basis of live weight, were cannulated in the abomasum, in the proximal jejunum approximately 4 m distal to the pylorus and in the terminal ileum. Six were infected with 3000 Trichostrongylus colubriformis and 3000 Ostertagia circumcincta larvae each day for 18 weeks and the remainder were pair-fed to individual infected lambs. All animals were offered ryegrass (Lolium perenne)–white clover (Trifolium repens) pasture, cut daily. Dry matter (DM) intake, live weight, faecal egg concentration, plasma pepsinogen and plasma protein concentrations were measured weekly. During weeks 7 and 17 after commencement of infection, the flow of digesta along the gastrointestinal tract was measured together with enteric plasma loss and true digestion and absorption of 125I-labelled albumin in the small intestine. DM intake was depressed by parasitism, being 1331, (se 70), 423 (se 32) and 529 (se 52) g/d during weeks 3, 7 and 17 respectively. The flow of nitrogen at the proximal jejunum and in faeces was increased by parasitism during week 7 and at the abomasum and ileum during week 17. Plasma protein-N loss (g/d) into the gastrointestinal tract was 0.68 (se 0.091) and 1.97 (se 0.139) during week 7, and 0.85 (se0.158) and 1.96 (se 0.396) during week 17, in control and infected sheep respectively. True digestion and absorption of albumin in the proximal small intestine, the site of infection, was very low (mean 0.08) and was not affected by parasitism. Between the abomasum and terminal ileum absorption of albumin was high (mean 0.87) and again was not affected by parasitism. It was calculated that of the total increase in endogenous protein passing from the ileum tract as a result of infection, plasma protein comprised only a small percentage (10–36%). The major proportion of digestion and absorption of protein occurred in the distal small intestine beyond the site of infection and was not affected by infection.
Large bowel fermentation in rats eating processed potatoes
Authors: Mathers J. C. , Dawson Lindsay D.
Published: May 2005

Four diets were prepared in which cooked potatoes provided approximately 0.8 of the dry matter (DM) and all the polysaccharides. The potatoes were either boiled conventionally (unprocessed) or prepared by rehydrating a commercial ‘instant’ potato product with boiling water. The diets were fed to rats (six per diet) immediately after preparation or after storage at 4° for 48 h and observations made on site and extent of digestion and large bowel (LB) fermentation. All diets were equally well digested (overall means 0.95 and 0.96 for DM and organic matter (OM) respectively) with a significant proportion of this digestion occurring in the LB. More OM flowed to the LB with the ‘instant’ (0.18 of intake) than with the unprocessed potato diets (0.13 of intake) and was associated with markedly different fermentation patterns. When compared with rats fed on the unprocessed potato diets, those given ‘instant’ potatoes had smaller caecums with much shorter transit times, lower caecal pH, higher total volatile fatty acid (VFA) concentrations and a greater molar proportion of butyrate in these VFA. Storage of the diets for 48 h at 4° had relatively smaller effects on these variables. Possible mechanisms for the observed effects and implications for tissue metabolism and health are discussed. In addition, a simple and apparently novel method for quantifying coprophagy is proposed.
Thermic effect of a meal
Authors: Piers L. S. , Soares M. J. , Shetty P. S.
Published: May 2005

The thermic effect of a standard liquid meal (TEM; energy 2.5 MJ; containing (g/kg) protein 100, fat 150, carbohydrate 750; volume 350 ml) was measured in a chronically undernourished (UN; n 9) group of human subjects and was compared with results from two control groups, one normal weight (NW)-for-height (BMI > 20; n 10) and the other underweight (UW)-for-height (BMI < 18, n 10), using a ventilated-hood system over a period of 6 h after ingestion of the meal. Results indicated that the UN subjects had lower values for body-weight, height, percentage fat and fat-free mass (FFM) compared with those of either control group. Basal metabolic rates were lowest in the UN group in absolute terms; however, there were no significant differences among groups on an analysis of covariance (ANACOVA) with FFM as the covariate. TEM responses in the UN group were significantly higher when expressed either in absolute terms or as a percentage of the energy density of the meal. The post-meal total energy output was significantly lower (P < 0.05) in the UW and UN groups as compared with the NW group in absolute terms; however, on adjusting for differences in FFM (by ANACOVA) there were no significant differences among groups. This would suggest that in the chronically undernourished thermogenic responses to a meal are unlikely to contribute towards any energy saving and may not constitute a part of any adaptive response to the undernourished state.

Chronic undernutrition: Thermic effect: Substrate oxidation rates: Body composition
Energy and nitrogen intake, expenditure and retention at 32° in growing fowl given diets with a wide range of energy and protein contents
Authors: Macleod M. G.
Published: May 2005

Heat production (HP) and the intake and retention of energy and nitrogen were measured in growing broiler fowl kept at 32° and given diets with metabolizable energy contents from 8 to 15 MJ/kg and crude protein (N × 6·25; CP) contents of 130 and 210 g/kg. The temperature of 32° was chosen for comparison with earlier measurements at 20° to minimize heat produced for the maintenance of body temperature. The effects of diet composition were observed when the same birds were taken from 20 to 32°. The tendency for energy intake to increase with dietary energy concentration was less at 32 than at 20°. The lower heat increments measured for the high-fat diets did not, therefore, confer an increased ability to sustain higher energy intake at 32°. HP was about 17% lower at 32 than at 20°; the change in HP between 20 and 32° was not significantly influenced by diet composition. The absence of significant effects of diet composition on HP, combined with the significant trend in energy intake, produced significant differences (related both to dietary energy and dietary protein concentrations) in total energy retention and in the partition of retained energy between protein and fat. As at 20°, variation in energy retention and in the composition of retained energy were the main responses to variation in dietary CP concentration and energy intake; a significantly higher energy cost of unit protein accretion on the low-CP diets was insufficient to produce an elevation in total HP because the higher unit energy cost was balanced by a lower absolute rate of protein accretion.
The influence of density and size of particles on rumination and passage from the reticulo-rumen of sheep
Authors: Kaske M. , Hatiboglu S. , ENGELHARDT W. V.
Published: May 2005

Plastic particles with different densities (0.92, 1.03, 1.22 and 1.44 g/ml) and sizes (1, 10 and 20 mm) were introduced into the rumen of fistulated sheep kept on a roughage diet. The forestomach was emptied 12 and 24 h after the introduction of the particles, and the contents were replaced by the same amount of rumen contents without plastic particles. The proportions of particles which left the reticulo-rumen (RR) during the experimental period were determined by collecting the faeces during the following 5 d. Non-ruminated particles were separated from the dried RR contents and from the faeces. Large particles were ruminated independently of particle size and density within the investigated range. After 12 and 24 h, 59 and 81% respectively of the particles initially introduced were comminuted due to rumination. During the 12 h period about four times as many particles with a density of 1.44 g/ml passed from the RR into the omasum compared with particles with a density of 0.92 or 1.03 g/ml. Three to ten times more 1 mm particles were excreted than originally-large particles (10 and 20 mm). Particles introduced with an original size of 10 or 20 mm were recovered mostly comminuted in the faeces. In a further experiment the rumens of eight sheep were emptied and filled with a buffer solution. Plastic particles (10 g) of each length (1, 5, 10 and 20 mm; all with a density of 1.03 g/ml) were introduced into the ventral rumen. Sedimentation of particles was prevented by gassing the solution in the RR. Of the initially introduced particles, 31.9, 25.4, 12.7 and 1.5% of the 1, 5, 10 and 20 mm long particles respectively left the RR within 4 h. It is concluded that rumination of particles is independent of particle density and size within the tested range. The probability of particles leaving the RR increases with the higher particle density and with the smaller size. If particle sedimentation is prevented in the RR even 10 mm long particles can leave the RR in considerable amounts.

Particle size: Particle density: Passage: Rumination: Sheep
The effect of sucrose supplements on particle-associated carboxymethylcellulase (EC 3.2.1.4) and xylanase (EC 3.2.1.8) activities in cattle given grass-silage-based diet
Authors: Huhtanen Pekka , Khalili Hannele
Published: May 2005

Carboxymethylcellulase (EC3.2.1.4; CMCase) and xylanase (EC3.2.1.8) activities were assayed in rumen fluid and from microbes closely associated either with rumen particulate material or with feed particles incubated in nylon bags in the rumen of cattle. The cattle were fitted with a permanent rumen cannula and a simple ‘T’-piece duodenal cannula and were given four diets in a 4 × 4 Latin Square experiment. The basal diet (diet C) consisted of grass silage, barley and rapeseed meal (700, 240 and 60 g/kg total dry matter (DM)) given at the rate of 5·3 kg/d or supplemented with 1·0 kg sucrose/d given twice daily (diet S), twice daily with 0·25 kg sodium bicarbonate/d (diet B) or as a continuous intrarumen infusion (diet I). Giving sucrose supplements decreased CMCase and xylanase activities extracted from microbes associated with rumen particulate material or feed particles incubated in nylon bags as compared with diet C. Supplementation of the sucrose diet with sodium bicarbonate resulted in higher CMCase and xylanase activities than other sucrose diets (S and I). Particle-associated CMCase and xylanase activities were found to be very sensitive in detecting differences in the rumen environment and were related to changes in cell wall digestion. The activities were highly correlated with disappearance of DM and neutral-detergent fibre from nylon bags incubated in the rumen, rumen and total digestion of cell-wall carbohydrates and rumen pool size of cell-wall carbohydrates. It was concluded that the attachment of fibrinolytic enzymes is involved in the depression of fibre digestion. Particle-associated CMCase and xylanase activities were much higher when measured from rumen particulate material than from feed particles incubated in nylon bags.
Dietary-induced increases of disaccharidase activities in rat jejunum
Authors: Santos Betty K. Samulitis-Dos , Goda Toshinao , Koldovsky Otakar
Published: May 2005

A study was carried out to examine whether the responsiveness of small intestinal epithelial cells to dietary carbohydrate varied during the daily 24 h cycle. The effect of sucrose on disaccharidase activities was compared during a period of decreasing disaccharidase activities, i.e. between 22.00 and 10.00 hours, and increasing disaccharidase activities, i.e. between 10.00 and 22.00 hours, in the jejunum of 7-week-old-rats. Rats were fed on a low-starch, high-fat diet (Lst; starch 5 and fat 73% of gross energy), or a high-starch, low-fat diet (Hst; starch 70 and fat 7% of gross energy). Both dietary groups exhibited typical diurnal variations in jejunal sucrase (EC3.2.1.48), maltase (EC3.2.1.20) and lactase (EC3.2.1.23) activities, exhibiting a peak around 22.00 hours and a trough at approximately 10.00 hours. When rats were fed on diet Lst for 7 d and then force-fed on an isoenergetic sucrose diet (S; sucrose 40 and fat 37% of gross energy) for 6 or 12 h they exhibited increased sucrase, maltase and lactase activities compared with rats fed on diet Lst. The absolute increase in disaccharidase activities was similar regardless of the time diet S was given or whether rats were killed at 10.00 hours or at 22.00 hours. Analyses of sucrase and lactase activities along the villus–crypt columns showed that the distribution of cell cohorts that responded to diet S was not influenced by the time of introduction of diet S. These findings suggest that small intestinal epithelial cells possess the ability to respond to dietary carbohydrate throughout the daily 24 h cycle.
Effects of dietary fibre and tannins from apple pulp on the composition of faeces in rats
Authors: Bravo L. , Saura-Calixto F. , Goni I.
Published: May 2005

The present study was undertaken to explore the effect of apple pulp on weight and composition of faeces. This material is rich in dietary fibre (DF;620 g dry matter/kg) and contains appreciable amounts of polyphenols. Recent reports indicate that both condensed tannins (CT) and soluble polyphenols form cross-links with protein and inhibit digestive enzymes, affecting the protein digestibility, and may produce a stimulation of endogenous nitrogen excretion. Two groups of male Wistar rats were fed on either a control diet free of DF or a diet containing 100 g apple pulp DF/kg during 7 d after a 4 d adaptation period. Body-weight and food intake were monitored daily and faeces and urine were collected once daily. DF, water content and polyphenolic compounds were measured in faeces, and N content in both faeces and urine. Faecal weight increased in the fibre group by 280 and 240% when compared with wet and dry faecal weights of animals fed on the fibre-free diet. Soluble dietary fibre (SDF) excreted in faeces was 10.9% of the SDF ingested, which suggested a low resistance to fermentation of this fraction. Of the insoluble DF, 43% of the ingested fibre was fermented. Polyphenols were degraded in the intestinal tract. Of the ingested CT, 68.6% was recovered in faeces, while the soluble polyphenols were extensively degraded (85.7% of that ingested). On the other hand, a higher faecal N excretion was observed for the fibre-fed group, suggesting a decrease in the digestibility of the dietary protein and lower apparent digestibility and N balance indices.
Role of the Septin Ring in the Asymmetric Localization of Proteins at the Mother-Bud Neck inSaccharomyces cerevisiae
Authors: Kozubowski Lukasz , Larson Jennifer R. , Tatchell Kelly
Published: May 2005

In the yeast Saccharomyces cerevisiae, septins form a scaffold in the shape of a ring at the future budding site that rearranges into a collar at the mother-bud neck. Many proteins bind asymmetrically to the septin collar. We found that the protein Bni4-CFP was located on the exterior of the septin ring before budding and on the mother side of the collar after budding, whereas the protein kinase Kcc4-YFP was located on the interior of the septin ring before budding and moved into the bud during the formation of the septin collar. Unbudded cells treated with the actin inhibitor latrunculin-A assembled cortical caps of septins on which Bni4-CFP and Kcc4-YFP colocalized. Bni4-CFP and Kcc4-YFP also colocalized on cortical caps of septins found in strains deleted for the genes encoding the GTPase activating proteins of Cdc42 (RGA1, RGA2, and BEM3). However, Bni4-CFP and Kcc4-YFP were still partially separated in mutants (gin4, elm1, cla4, and cdc3-1) in which septin morphology was severely disrupted in other ways. These observations provide clues to the mechanisms for the asymmetric localization of septin-associated proteins.
Atg17 Regulates the Magnitude of the Autophagic Response
Authors: Cheong Heesun , Yorimitsu Tomohiro , Reggiori Fulvio , Legakis Julie E. , Wang Chao-Wen , Klionsky Daniel J.
Published: May 2005

Autophagy is a catabolic process used by eukaryotic cells for the degradation and recycling of cytosolic proteins and excess or defective organelles. In yeast, autophagy is primarily a response to nutrient limitation, whereas in higher eukaryotes it also plays a role in developmental processes. Due to its essentially unlimited degradative capacity, it is critical that regulatory mechanisms are in place to modulate the timing and magnitude of the autophagic response. One set of proteins that seems to function in this regard includes a complex that contains the Atg1 kinase. Aside from Atg1, the proteins in this complex participate primarily in either nonspecific autophagy or specific types of autophagy, including the cytoplasm to vacuole targeting pathway, which operates under vegetative growth conditions, and peroxisome degradation. Accordingly, these proteins are prime candidates for factors that regulate the conversion between these pathways, including the change in size of the sequestering vesicle, the most obvious morphological difference. The atg17Δ mutant forms a reduced number of small autophagosomes. As a result, it is defective in peroxisome degradation and is partially defective for autophagy. Atg17 interacts with both Atg1 and Atg13, via two coiled-coil domains, and these interactions facilitate its inclusion in the Atg1 complex.
A human splicing factor, SKIP, associates with P-TEFb and enhances transcription elongation by HIV-1 Tat
Authors: Brès Vanessa , Gomes Nathan , Pickle Loni , Jones Katherine A.
Published: May 2005

HIV-1 Tat binds human CyclinT1 and recruits the CDK9/P-TEFb complex to the viral TAR RNA in a step that links RNA polymerase II (RNAPII) C-terminal domain (CTD) Ser 2 phosphorylation with transcription elongation. Previous studies have suggested a connection between Tat and pre-mRNA splicing factors. Here we show that the splicing-associated c-Ski-interacting protein, SKIP, is required for Tat transactivation in vivo and stimulates HIV-1 transcription elongation, but not initiation, in vitro. SKIP associates with CycT1:CDK9/P-TEFb and Tat:P-TEFb complexes in nuclear extracts and interacts with recombinant Tat:P-TEFb:TAR RNA complexes in vitro, indicating that it may act through nascent RNA to overcome pausing by RNAPII. SKIP also associates with U5snRNP proteins and tri-snRNP110K in nuclear extracts, and facilitates recognition of an alternative Tat-specific splice site in vivo. The effects of SKIP on transcription elongation, binding to P-TEFb, and splicing are mediated through the SNW domain. HIV-1 Tat transactivation is accompanied by the recruitment of P-TEFb, SKIP, and tri-snRNP110K to the integrated HIV-1 promoter in vivo, whereas the U5snRNPs associate only with the transcribed coding region. These findings suggest that SKIP plays independent roles in transcription elongation and pre-mRNA splicing.
Proapoptotic Bak is sequestered by Mcl-1 and Bcl-xL, but not Bcl-2, until displaced by BH3-only proteins
Authors: Willis Simon N. , Chen Lin , Dewson Grant , Wei Andrew , Naik Edwina , Fletcher Jamie I. , Adams Jerry M. , Huang David C.S.
Published: May 2005

Commitment of cells to apoptosis is governed largely by the interaction between members of the Bcl-2 protein family. Its three subfamilies have distinct roles: The BH3-only proteins trigger apoptosis by binding via their BH3 domain to prosurvival relatives, while the proapoptotic Bax and Bak have an essential downstream role involving permeabilization of organellar membranes and induction of caspase activation. We have investigated the regulation of Bak and find that, in healthy cells, Bak associates with Mcl-1 and Bcl-xL but surprisingly not Bcl-2, Bcl-w, or A1. These interactions require the Bak BH3 domain, which is also necessary for Bak dimerization and killing activity. When cytotoxic signals activate BH3-only proteins that can engage both Mcl-1 and Bcl-xL (such as Noxa plus Bad), Bak is displaced and induces cell death. Accordingly, the BH3-only protein Noxa could bind to Mcl-1, displace Bak, and promote Mcl-1 degradation, but Bak-mediated cell death also required neutralization of Bcl-xL by other BH3-only proteins. The results indicate that Bak is held in check solely by Mcl-1 and Bcl-xL and induces apoptosis only if freed from both. The finding that different prosurvival proteins have selective roles has notable implications for the design of anti-cancer drugs that target the Bcl-2 family.
Histone deposition protein Asf1 maintains DNA replisome integrity and interacts with replication factor C
Authors: Franco Alexa A. , Lam Wendy M. , Burgers Peter M. , Kaufman Paul D.
Published: May 2005

Chromatin assembly and DNA replication are temporally coupled, and DNA replication in the absence of histone synthesis causes inviability. Here we demonstrate that chromatin assembly factor Asf1 also affects DNA replication. In budding yeast cells lacking Asf1, the amounts of several DNA replication proteins, including replication factor C (RFC), proliferating cell nuclear antigen (PCNA), and DNA polymerase ϵ (Pol ϵ), are reduced at stalled replication forks. In contrast, DNA polymerase α (Pol α) accumulates to higher than normal levels at stalled forks in asf1Δ cells. Using purified, recombinant proteins, we demonstrate that RFC directly binds Asf1 and can recruit Asf1 to DNA molecules in vitro. We conclude that histone chaperone protein Asf1 maintains a subset of replication elongation factors at stalled replication forks and directly interacts with the replication machinery.
AtPDR12 Contributes to Lead Resistance in Arabidopsis
Authors: Lee Miyoung , Lee Kiyoul , Lee Joohyun , Noh Eun Woon , Lee Youngsook
Published: May 2005

Arabidopsis (Arabidopsis thaliana) contains about 130 ATP-binding cassette (ABC) proteins, which are likely to contribute to the transport of diverse materials, including toxic substances. However, the substrates of ABC transporters remain unknown in most cases. We tested which ABC transporter is involved in detoxification of lead [Pb(II)]. Among the many tested, we found that the message level of only AtPDR12 increased in both shoots and roots of Pb(II)-treated Arabidopsis, suggesting that it may be involved in the detoxification of Pb(II). AtPDR12-knockout plants (atpdr12) were used to further test this possibility. In Pb(II)-containing medium, atpdr12 plants grew less well and had higher Pb contents than those of wild-type plants. In contrast, AtPDR12-overexpressing Arabidopsis plants were more resistant to Pb(II) and had lower Pb contents than wild-type plants. The mutant phenotypes and their Pb contents, as well as the localization of the GFP:AtPDR12 fusion protein at the plasma membrane, suggest that AtPDR12 functions as a pump to exclude Pb(II) and/or Pb(II)-containing toxic compounds from the cytoplasm. Inhibition of glutathione synthesis by addition of buthionine sulfoximine to the growth medium exacerbated the Pb(II)-sensitive phenotype of atpdr12 plants, consistent with a glutathione-dependent detoxification mechanism operating in parallel with an AtPDR12-dependent mechanism. Thus, we propose that AtPDR12 is an ABC transporter that contributes to Pb(II) resistance in Arabidopsis.
The Role of Salicylic Acid in the Induction of Cell Death in Arabidopsis acd11
Authors: Brodersen Peter , Malinovsky Frederikke Gro , Hématy Kian , Newman Mari-Anne , Mundy John
Published: May 2005

Salicylic acid (SA) is implicated in the induction of programmed cell death (PCD) associated with pathogen defense responses because SA levels increase in response to PCD-inducing infections, and PCD development can be inhibited by expression of salicylate hydroxylase encoded by the bacterial nahG gene. The acd11 mutant of Arabidopsis (Arabidopsis thaliana L. Heynh.) activates PCD and defense responses that are fully suppressed by nahG. To further study the role of SA in PCD induction, we compared phenotypes of acd11/nahG with those of acd11/eds5-1 and acd11/sid2-2 mutants deficient in a putative transporter and isochorismate synthase required for SA biosynthesis. We show that sid2-2 fully suppresses SA accumulation and cell death in acd11, although growth inhibition and premature leaf chlorosis still occur. In addition, application of exogenous SA to acd11/sid2-2 is insufficient to restore cell death. This indicates that isochorismate-derived compounds other than SA are required for induction of PCD in acd11 and that some acd11 phenotypes require NahG-degradable compounds not synthesized via isochorismate.
Mutations in the Arabidopsis Phosphoinositide Phosphatase Gene SAC9 Lead to Overaccumulation of PtdIns(4,5)P2 and Constitutive Expression of the Stress-Response Pathway
Authors: Williams Mary Elizabeth , Torabinejad Javad , Cohick Evan , Parker Katherine , Drake Elizabeth J. , Thompson James E. , Hortter Michelle , DeWald Daryll B.
Published: May 2005

Phosphoinositides (PIs) are signaling molecules that regulate cellular events including vesicle targeting and interactions between membrane and cytoskeleton. Phosphatidylinositol (PtdIns)(4,5)P2 is one of the best characterized PIs; studies in which PtdIns(4,5)P2 localization or concentration is altered lead to defects in the actin cytoskeleton and exocytosis. PtdIns(4,5)P2 and its derivative Ins(1,4,5)P3 accumulate in salt, cold, and osmotically stressed plants. PtdIns(4,5)P2 signaling is terminated through the action of inositol polyphosphate phosphatases and PI phosphatases including supressor of actin mutation (SAC) domain phosphatases. In some cases, these phosphatases also act on Ins(1,4,5)P3. We have characterized the Arabidopsis (Arabidopsis thaliana) sac9 mutants. The SAC9 protein is different from other SAC domain proteins in several ways including the presence of a WW protein interaction domain within the SAC domain. The rice (Oryza sativa) and Arabidopsis SAC9 protein sequences are similar, but no apparent homologs are found in nonplant genomes. High-performance liquid chromatography studies show that unstressed sac9 mutants accumulate elevated levels of PtdIns(4,5)P2 and Ins(1,4,5)P3 as compared to wild-type plants. The sac9 mutants have characteristics of a constitutive stress response, including dwarfism, closed stomata, and anthocyanin accumulation, and they overexpress stress-induced genes and overaccumulate reactive-oxygen species. These results suggest that the SAC9 phosphatase is involved in modulating phosphoinsitide signals during the stress response.
Heat Stress Phenotypes of Arabidopsis Mutants Implicate Multiple Signaling Pathways in the Acquisition of Thermotolerance
Authors: Larkindale Jane , Hall Jennifer D. , Knight Marc R. , Vierling Elizabeth
Published: May 2005

To investigate the importance of different processes to heat stress tolerance, 45 Arabidopsis (Arabidopsis thaliana) mutants and one transgenic line were tested for basal and acquired thermotolerance at different stages of growth. Plants tested were defective in signaling pathways (abscisic acid, salicylic acid, ethylene, and oxidative burst signaling) and in reactive oxygen metabolism (ascorbic acid or glutathione production, catalase) or had previously been found to have temperature-related phenotypes (e.g. fatty acid desaturase mutants, uvh6). Mutants were assessed for thermotolerance defects in seed germination, hypocotyl elongation, root growth, and seedling survival. To assess oxidative damage and alterations in the heat shock response, thiobarbituric acid reactive substances, heat shock protein 101, and small heat shock protein levels were determined. Fifteen mutants showed significant phenotypes. Abscisic acid (ABA) signaling mutants (abi1 and abi2) and the UV-sensitive mutant, uvh6, showed the strongest defects in acquired thermotolerance of root growth and seedling survival. Mutations in nicotinamide adenine dinucleotide phosphate oxidase homolog genes (atrbohB and D), ABA biosynthesis mutants (aba1, aba2, and aba3), and NahG transgenic lines (salicylic acid deficient) showed weaker defects. Ethylene signaling mutants (ein2 and etr1) and reactive oxygen metabolism mutants (vtc1, vtc2, npq1, and cad2) were more defective in basal than acquired thermotolerance, especially under high light. All mutants accumulated wild-type levels of heat shock protein 101 and small heat shock proteins. These data indicate that, separate from heat shock protein induction, ABA, active oxygen species, and salicylic acid pathways are involved in acquired thermotolerance and that UVH6 plays a significant role in temperature responses in addition to its role in UV stress.
The Arabidopsis Plastidic Methionine Sulfoxide Reductase B Proteins. Sequence and Activity Characteristics, Comparison of the Expression with Plastidic Methionine Sulfoxide Reductase A, and Induction by Photooxidative Stress
Authors: Vieira Dos Santos Christina , Cuiné Stéphan , Rouhier Nicolas , Rey Pascal
Published: May 2005

Two types of methionine (Met) sulfoxide reductases (Msr) catalyze the reduction of Met sulfoxide (MetSO) back to Met. MsrA, well characterized in plants, exhibits an activity restricted to the Met-S-SO-enantiomer. Recently, a new type of Msr enzyme, called MsrB, has been identified in various organisms and shown to catalytically reduce the R-enantiomer of MetSO. In plants, very little information is available about MsrB and we focused our attention on Arabidopsis (Arabidopsis thaliana) MsrB proteins. Searching Arabidopsis genome databases, we have identified nine open reading frames encoding proteins closely related to MsrB proteins from bacteria and animal cells. We then analyzed the activity and abundance of the two chloroplastic MsrB proteins, MsrB1 and MsrB2. Both enzymes exhibit an absolute R-stereospecificity for MetSO and a higher catalytic efficiency when using protein-bound MetSO as a substrate than when using free MetSO. Interestingly, we observed that MsrB2 is reduced by thioredoxin, whereas MsrB1 is not. This feature of MsrB1 could result from the lack of the catalytical cysteine (Cys) corresponding to Cys-63 in Escherichia coli MsrB that is involved in the regeneration of Cys-117 through the formation of an intramolecular disulfide bridge followed by thioredoxin reduction. We investigated the abundance of plastidial MsrA and B in response to abiotic (water stress, photooxidative treatment) and biotic (rust fungus) stresses and we observed that MsrA and B protein levels increase in response to the photooxidative treatment. The possible role of plastidic MsrB in the tolerance to oxidative damage is discussed.
Constitutively High Expression of the Histidine Biosynthetic Pathway Contributes to Nickel Tolerance in Hyperaccumulator Plants
Authors: Ingle Robert A. , Mugford Sam T. , Rees Jonathan D. , Campbell Malcolm M. , Smith J. Andrew C.
Published: May 2005

Plants that hyperaccumulate Ni exhibit an exceptional degree of Ni tolerance and the ability to translocate Ni in large amounts from root to shoot. In hyperaccumulator plants in the genus Alyssum, free His is an important Ni binding ligand that increases in the xylem proportionately to root Ni uptake. To determine the molecular basis of the His response and its contribution to Ni tolerance, transcripts representing seven of the eight enzymes involved in His biosynthesis were investigated in the hyperaccumulator species Alyssum lesbiacum by RNA gel blot analysis. None of the transcripts changed in abundance in either root or shoot tissue when plants were exposed to Ni, but transcript levels were constitutively higher in A. lesbiacum than in the congeneric nonaccumulator A. montanum, especially for the first enzyme in the biosynthetic pathway, ATP-phosphoribosyltransferase (ATP-PRT). Comparison with the weak hyperaccumulator A. serpyllifolium revealed a close correlation between Ni tolerance, root His concentration, and ATP-PRT transcript abundance. Overexpression of an A. lesbiacum ATP-PRT cDNA in transgenic Arabidopsis thaliana increased the pool of free His up to 15-fold in shoot tissue, without affecting the concentration of any other amino acid. His-overproducing lines also displayed elevated tolerance to Ni but did not exhibit increased Ni concentrations in either xylem sap or shoot tissue, suggesting that additional factors are necessary to recapitulate the complete hyperaccumulator phenotype. These results suggest that ATP-PRT expression plays a major role in regulating the pool of free His and contributes to the exceptional Ni tolerance of hyperaccumulator Alyssum species.
AUXIN RESPONSE FACTOR7 Restores the Expression of Auxin-Responsive Genes in Mutant Arabidopsis Leaf Mesophyll Protoplasts
Authors: Wang Shucai , Tiwari Shiv B. , Hagen Gretchen , Guilfoyle Tom J.
Published: May 2005

AUXIN RESPONSE FACTOR7 (ARF7) is one of five ARF transcriptional activators in Arabidopsis thaliana that is proposed to regulate auxin-responsive expression of genes containing TGTCTC auxin response elements in their promoters. An Arabidopsis mutant (nonphototropic hypocotyl4-1 [nph4-1]) that is a null for ARF7 showed strongly reduced expression of integrated auxin-responsive reporter genes and natural genes that were monitored in Arabidopsis leaf mesophyll protoplasts. Expression of the reporter and natural genes was restored in an auxin-dependent manner when protoplasts were transfected with a 35S:ARF7 effector gene, encoding a full-length ARF7 protein. Transfection of effector genes encoding other ARF activators restored auxin-responsive gene expression to varying degrees, but less than that observed with the ARF7 effector gene. Arabidopsis lines that were null for ARF6, ARF8, or ARF19 were not defective in expression of the reporter and natural auxin response genes assayed in mesophyll protoplasts, suggesting that ARF7 plays a major role in regulating expression of a subset of auxin response genes in leaf mesophyll cells. Auxin-responsive gene expression was induced in wild-type protoplasts and restored in nph4-1 protoplasts only with auxin and not with other hormones, including brassinolide. In the presence of auxin, however, brassinolide modestly enhanced auxin-responsive gene expression.
A Conserved Transcription Factor Mediates Nuclear Control of Organelle Biogenesis in Anciently Diverged Land Plants
Authors: Yasumura Yuki , Moylan Elizabeth C. , Langdale Jane A.
Published: May 2005

Land plant chloroplasts evolved from those found in the green algae. During land plant evolution, nuclear regulatory mechanisms have been modified to produce morphologically and functionally diverse chloroplasts in distinct developmental contexts. At least some of these mechanisms evolved independently in different plant lineages. In angiosperms, GOLDEN2-LIKE (GLK) transcription factors regulate the development of at least three chloroplast types. To determine whether GLK-mediated regulation of chloroplast development evolved within angiosperms or is a plesiomorphy within land plants, gene function was examined in the moss Physcomitrella patens. Gene expression patterns and loss-of-function mutant phenotypes suggested that GLK gene function is conserved between P. patens and Arabidopsis thaliana, species that diverged >400 million years ago. In support of this suggestion, moss genes partially complement Arabidopsis loss-of-function mutants. Therefore, GLK-mediated regulation of chloroplast development defines one of the most ancient conserved regulatory mechanisms identified in the plant kingdom.
Genetic linkage between Polι deficiency and increased susceptibility to lung tumors in mice
Authors: Lee Gang‐Hong , Matsushita Hiroshi
Published: May 2005

Polι is a member of the Y‐family DNA polymerases, characterized by their capacity for translesion DNA synthesis and low fidelity base incorporation, and has therefore been assumed to play important roles in mutagenesis and carcinogenesis. In fact, the mouse Polι gene is located within the Par2 (pulmonary adenoma resistance 2) locus on distal chromosome 18, which we have identified as a major susceptibility locus regarding urethane induction of pulmonary adenomas. Indeed, Polι has been suggested to be a candidate for Par2 from both the genetic and biological standpoints. Taking advantage of 129X1/SvJ mice naturally deficient in Polι due to a nonsense mutation within the coding region of the gene, we here analyzed urethane‐treated (A/J × 129X1/SvJ)F1 × A/J backcross and (A/J × 129X1/SvJ)F2 intercross mice and observed the defective 129X1/SvJ Polι allele to be genetically linked with an increased susceptibility to lung tumors relative to the A/J allele. Thus, among the already known mouse Polι alleles, the defective 129X1/SvJ allele is associated exclusively with the highest susceptibility to lung tumors. The result indicates a possibility that the Polι gene may participate in error‐free repair of damaged DNA and prevention of lung tumor development. (Cancer Sci 2005; 96: 256 –259)
A tumor‐specific gene therapy strategy targeting dysregulation of the VHL/HIF pathway in renal cell carcinomas
Authors: Ogura Masakazu , Shibata Toru , Yi Junlin , Liu Junye , Qu Runjiang , Harada Hiroshi , Hiraoka Masahiro
Published: May 2005

Hypoxia‐inducible factors, key transcription factors for hypoxia‐dependent gene expression, play important roles in angiogenesis and tumor growth. The VHL protein binds to the α subunit of (HIF‐α) for its oxygen‐dependent degradation. VHL mutations are found frequently in sporadic RCC. Disruption of VHL results in an abnormal accumulation of HIF‐α, leading to the upregulation of downstream genes such as the vascular endothelial growth factor gene. We constructed a luciferase reporter vector driven by hypoxia‐responsive elements (5HRE/luc) and a therapeutic vector expressing a herpes simplex virus thymidine kinase gene (5HRE/tk). In the transient transfection assay using VHL‐deficient 786‐O cells, constitutive luciferase expression was detected under both aerobic and hypoxic conditions. In contrast, 786‐O cells transfected with a wild‐type VHL showed hypoxia‐inducible luciferase activity. In in vitro MTS assay, 50% of growth inhibition of 786‐O cells stably transfected with 5HRE/tk was achieved with exposure to 0.2 µg/mL of GCV under both aerobic and hypoxic conditions. Xenografts of the stable clone in SCID mice exhibited a marked regression on daily injections of GCV (50 mg/kg) for 10 days. In conclusion, a hypoxia‐responsive vector may have therapeutic potential for RCC with VHL mutations. (Cancer Sci 2005; 96: 288 –294)