bluuebunny/crossref_metadata_2025 huggingface dataset:
Production of senescent cells of Saccharomyces cerevisiae by centrifugal elutriation
Authors: Woldringh C. L. , Fluiter K. , Huls P. G.
Published: May 2005

The centrifugal elutriator has been used as a baby machine by loading the chamber with a population of mixed‐generation daughter cells and allowing this population to grow, divide and age under continuous washing‐out of newborn daughter cells. Clear peaks in the number of elutriated cells were reproducibly obtained for at least ten generations. The parent cells growing in the chamber continued to divide at the steady‐state generation time of 95–100 min, showing no change in cycle time during aging. The washed‐out daughter cells increased in volume during the first five generations from their steady‐state value of 17 μm3 to a maximum of 34 μm3. As to be expected, the generation times of these large daughters, determined in a synchronous batch culture, were shorter (130 min) than that of the steady‐state daughters (240 min), even when derived from 15‐generation parents. No indication for a volume increase of daughter cells without bud was observed when a population was allowed to grow in the chamber without washing‐out the smaller daughter cells. The 15‐generation parent population, recovered from the chamber, had an average volume of 80 μm3 and consisted of: (i) 71% cells with more than ten scars, (ii) 13% cells with one to nine scars, and (iii) 17% daughter cells. The production of senescent cells by undisturbed growth in the elutriator chamber has been prolonged to 29 generations. The method is therefore suitable to examine what factors determine the life span of budding yeast.
The complete sequence of a 9037 bp DNA fragment of the right arm of Saccharomyces cerevisiae chromosome VII
Authors: Arroyo Javier , García‐Gonzalez Melba , García‐Saez M. Isabel , Sánchez Miguel , Nombela César
Published: May 2005

We report the sequence of a 9037 bp fragment from the right arm of Saccharomyces cerevisiae chromosome VII. Analysis of the sequence revealed four complete open reading frames (ORFs), namely G7572, G7576, G7579 and G7584. The first three corresponded, respectively, to the previously cloned genes: HIP1, coding for a high‐affinity histidine‐specific permease, TDH1, one of the known genes coding for glyceraldehyde‐3‐phosphate dehydrogenase and ODPX, which encodes a precursor of protein X, a component of the pyruvate dehydrogenase complex. The ORF G7584 showed 35·8% identity with a hypothetical protein of Caenorhabditis elegans chromosome 3. The reported sequence has been deposited in the EMBL data library under Accession Number X82408.
Two‐Dimensional protein map of Saccharomyces cerevisiae: Construction of a gene–protein index
Authors: Boucherie Helian , Monribot Christelle , Perrot Michel , Dujardin Genevieve , Kermorgant Michele , Slonimski Piotr
Published: May 2005

This publication marks the beginning of the construction of a gene–protein index that relates proteins which are resolved on the two‐dimensional protein map of Saccharomyces cerevisiae with their corresponding genes. We report the identification of 36 novel polypeptide spots on the yeast protein map. They correspond to the products of 26 genes. Together with the polypeptide spots previously identified, this raises to 41 the number of genes whose products have been identified on the protein map. The proteins identified here are concerned with four major areas of yeast cellular physiology: carbon metabolism, heat shock, amino acid biosynthesis and purine biosynthesis. Given the molecular weight and isoelectric point of the identified proteins, and the codon‐usage bias of the corresponding genes, it can be estimated that 25 to 35% of all the soluble yeast proteins are detectable under the labelling and running gel conditions used in this study.
In vivo cloning by homologous recombination in yeast using a two‐plasmid‐based system
Authors: Degryse Eric , Dumas Bruno , Dietrich Mireille , Laruelle Laurence , Achstetter Tilman
Published: May 2005

In order to reduce the number of classical DNA manipulation and ligation steps in the generation of yeast expression plasmids, a series of vectors is described which facilitate the assembly of such plasmids by the more efficient ‘recombination in vivo’ technique. Two sets of vectors were developed. The first set, called ‘expression vectors’, contains an expression cassette with a yeast promoter and the PGK terminator separated by a polylinker, and an Escherichia coli replicon. Subcloning in these vectors of a DNA fragment generates a ‘transfer vector’ which is compatible with the second set of E. coli–yeast shuttle vectors. This set of ‘recombination vectors’ contains a cassette for a functional copy of a gene complementing a host strain auxotrophy or a bacterial gene conferring an antibiotic resistance to the plasmid‐bearing host. Plasmid copy numbers can be modulated through the use of URA3 or URA3‐d as the selective marker together with an ARS/CEN and the 2 μm replicon.

Integration of the cloned DNAs into the yeast linearized replicative vectors occurs by recombination between homologous flanking sequences during transformation in yeast or E. coli. All the vectors contain the origin of replication of phage f1 and allow the generation of single‐stranded DNA in E. coli for sequencing or site‐directed mutagenesis.

The sequence presented (Figure 1a) has been entered in the EMBL data library under Accession Number Z48747.
Host cell properties and external pH affect proinsulin production by Saccharomyces yeast
Authors: Kozlov Dmitry G. , Prahl Natalija , Efremov Boris D. , Peters Lars , Benevolensky Sergei V. , Wambut Rolf , Karpychev Igor V. , Eldarov Mikhail A.
Published: May 2005

The expression of a hybrid gene encoding an α‐factor prepro leader peptide‐miniproinsulin (MPI) fusion [MPI is the same as the LysArg human insulin precursor described by Thim et al. (1986)] was tested in a series of isogenic yeast strains to investigate the influence of some genetic and physiological factors on heterologous production in yeast. We found that: (i) an MFα1 gene disruption in haploid cells, as well as MFα1 gene product expression in diploid cells, do not affect the MPI secretion level; (ii) under conditions of exogenous leucine availability, MPI production is hindered by leucine auxotrophy (a leu2 mutation); (iii) rho− mutations increase the per‐cell MPI yield approximately three‐fold; (iv) the MPI yield is apparently dependent on the pH of the culture medium: the higher the external pH, the larger the per‐cell MPI yield.
Sequence, map position and genome organization of the RPL17B gene, encoding ribosomal protein L17b in Saccharomyces cerevisiae
Authors: Berroteran Rhonda W. , Hampsey Michael
Published: May 2005

Sequence analysis of the newly defined SSU81 gene revealed an adjacent open reading frame (ORF) encoding a protein whose deduced amino acid sequence is identical to that of ribosomal protein L17. The DNA sequence of this region is different from that of the RPL17A gene and therefore represents a duplicate gene encoding L17. We have designated this gene RPL17B. The RPL17B coding region is split by an intron that occurs in the same position (codons 14/15) as the intron in RPL17A. The RPL17B promoter region includes two TATA boxes, a canonical UASRPG motif, and several pyrimidine‐rich tracts. RPL17B was mapped by CHEF and lambda clone grid hybridization blots to the right arm of chromosome V, linked to the TRP2 and RAD51 genes. A partial ORF was identified adjacent to RPL17B and SSU81 that is homologous to an ORF (designated A509) physically linked to RPL17A. This observation, and the identical position of the introns within the RPL17 genes, suggest that one RPL17 locus arose by duplication and translocation of the other. The complete 3·8 kbp DNA sequence encompassing RPL17B has been entered in the GenBank data library under Accession Number U15653.
The Saccharomyces cerevisiae FLO1 flocculation gene encodes for a cell surface protein
Authors: Bidard Frederique , Bony Muriel , Blondin Bruno , Dequin Sylvie , Barre Pierre
Published: May 2005

The sequencing of a 6619 bp region encoding for a flocculation gene previously cloned from a strain defined as FLO5 (Bidard et al., 1994) has revealed that it was a FLO1 gene. The FLO1 gene product has been localized at the cell surface of the yeast cell by immunofluorescent microscopy. The Flo1 protein contains four regions with repeated sequences which account for about 70% of the amino acids of this protein. A functional analysis of the major repeated region has revealed that it plays an important role in determining the flocculation level. A gene disruption experiment has shown that the FLO5 strain STX 347‐1D contains at least two flocculation genes of the FLO1 type but that they are supposed to be inactive and do not contribute to its flocculation. However, enzyme‐linked immunosorbent assays performed on intact cells have revealed that a protein expressed at the cell surface of the FLO5 strain STX 347‐1D is antigenically related to Flo1p. A deletion analysis of the 5′ region of the FLO1 gene has shown that the expression is submitted to controls which depend on the genetic background of the strain.
A new essential gene located on Saccharomyces cerevisiae chromosome IX
Authors: Kurlandzka Anna , Rytka Joanna , Gromadka Robert , Murawski Marcin
Published: May 2005

A new 1150 amino acids long open reading frame (ORF), coding for an essential protein of unknown function was found Saccharomyces cerevisiae by sequencing 3754 bp of geonomic DNA. The clone was isolated in a search for a fatty acid‐binding protein (FABP) and was localized on chromosome IX. The ORF bears no homology to FABP, but it shows weak similarity to Plasmodium vivax reticulocyte binding protein 1 and to aggregation‐specific adenylate cyclase from Dictyostelium discoideum. The new gene is constitutively transcribed regardless of the carbon source used. The nucleotide sequence reported in this paper has been deposited in GenBank (Accession Number U17918).
Improvement of chromosomal DNA extraction from different yeast species by analysis of single preparation steps
Authors: Cardinali Gianluigi , Pellegrini Leandro , Martini Alessandro
Published: May 2005

A modified procedure is proposed for chromosomal DNA extraction based on a cell‐wall lytic enzyme never applied before in pulsed field gel electrophoresis. Protoplasting efficiency is retained under very challenging conditions for enzyme activity, such as those required for non‐Saccharomyces yeasts often characterized by cell walls highly resistant to lysis.
XV. Yeast sequencing reports. Sequence analysis of a 9873 bp fragment of the left arm of yeast chromosome XV that contains the ARG8 and CDC33 genes, a putative riboflavin synthase beta chain gene, and four new open reading frames
Authors: Casas Celia , Aldea Marti , Herrero Enrique , Casamayor Antonio , Ariño Joaquin , Lafuente Maria Jose , Gamo Francisco‐Javier , Gancedo Carlos
Published: May 2005

The DNA sequence of a 9873 bp fragment located near the left telomere of chromosome XV has been determined. Sequence analysis reveals seven open reading frames. One is the ARG8 gene coding for N‐acetylornithine aminotransferase. Another corresponds to CDC33, which codes for the initiation factor 4E or cap binding protein. The open reading frame AOE169 can be considered as the putative gene for the Saccharomyces cerevisiae riboflavin synthase beta chain, since its translation product shows strong homology with four prokaryotic riboflavin synthase beta chains. The nucleotide sequence reported here has been submitted to the EMBL data library under the Accession Number X84036.
XV. Yeast sequencing reports. Sequence analysis of a 44 kb DNA fragment of yeast chromosome XV including the Ty1‐H3 retrotransposon, the suf1(+) frameshift suppressor gene for tRNA‐Gly, the yeast transfer RNA‐Thr‐1a and a delta element
Authors: Vandenbol Micheline , Durand Patrick , Portetelle Daniel , Hilger Francois
Published: May 2005

We have sequenced on both strands a 44,019 bp fragment located on the left arm of Saccharomyces cerevisiae chromosome XV.

The sequenced segment contains 22 open reading frames (ORFs) of at least 100 amino acids long, one of which probably contains an intron. Six of the 22 ORFs correspond to known proteins: the multicopy suppressor of Snf1 protein 1, the two Ty1‐H3 transposon proteins TyA and TyB, the myo‐inositol transporter 2, the transcription factor protein Ino4 and the 3,4‐dihydroxy‐5‐hexaprenylbenzoate methyltransferase. Of the 16 remaining ORFs, two show highest homologies with the yeast serine/threonine protein kinase Ste20 and the human tryptophanyl‐tRNA synthetase. Eight ORFs show slight similarities with protein sequences described in data banks.

DNA sequence comparison reveals also the presence of three known sequences: the Ty1‐H3 transposable element, the yeast suf1(+) frameshift suppressor gene for tRNA‐Gly and the yeast transfer RNA‐Thr‐1a. A fourth DNA sequence shows striking identities with the yeast delta elements.

The 44,019 bp sequence has been entered in the EMBL data library under Accession Number Z48149.
A disruption‐replacement approach for the targeted integration of foreign genes in Hansenula polymorpha
Authors: Agaphonov Michael O. , Beburov Michael Yu. , Ter‐Avanesyan Michael D. , Smirnov Vladimir N.
Published: May 2005

A system has been developed which allows the selection of integrative transformants with replacement of the Hansenula polymorpha methanol oxidase gene (MOX) with expression cassettes carrying heterologous gene under the control of the MOX promoter. The system is convenient for comparison of the expression levels of different constructs integrated into the same locus of the H. polymorpha genome. This system was used to compare the secretion levels of human urinary plasminogen activator, the secretion of which was directed by different signal sequences.
XV. Yeast sequencing reports. DNA sequence analysis of a 13 kbp fragment of the left arm of yeast chromosome XV containing seven new open reading frames
Authors: Casamayor Antonio , Aldea Martí , Casas Celia , Herrero Enrique , Gamo Francisco‐Javier , Lafuente María J. , Gancedo Carlos , Ariño Joaquín
Published: May 2005

The sequence of a 13 kbp fragment located in the vicinity of the left telomere of chromosome XV (cosmid pEOA179) has been determined. Seven new open reading frames (ORFs) encoding polypeptides longer than 100 residues have been found (AOB629, AOA342, AOC231, AOE555, AOE236, AOA236 and AOE1045). Three of them show no identity with proteins deposited in the data banks. ORF AOB629 (629 amino acids) has some similarity with previously described ferric reductases from Saccharomyces cerevisiae and Schizosaccharomyces pombe. ORF AOA342 encodes a polypeptide reminiscent of dihydroflavonol‐4‐reductases from a number of plant species. AOE236 displays a high level of identity when compared with peroxisomal membrane proteins previously cloned from the methylotrophic yeast Candida boidinii. Finally, AOE1045 encodes a large protein (1045 residues) with some identity with a hypothetical 147 kDa protein identified during the sequencing of Caenorhabditis elegans chromosome 3. The complete nucleotide sequence of the 13 kbp fragment has been deposited at the EMBL data base (Accession Number Z48239).
Yeast mapping reports. Clone bank of the mitochondrial genome of yeast Hansenula wingei
Authors: Sekito Takayuki , Okamoto Kozi , Kitano Hiromichi , Yoshida Kazuo
Published: May 2005

For sequencing, mitochondrial DNA from Hansenula wingei yeast was digested with various restriction enzymes and the resultant DNA fragments were cloned into a pEMBL phasmid vector. Our clone bank consists of 39 overlapping clones which cover the entire 27 694 bp region of the H. wingei mitochondrial genome. The nucleotide sequence data reported in this paper will appear in the DDBJ, EMBL and GenBank Nucleotide Sequence Database with the following Accession Number: D31785.
Further definition of the sequence and position requirements of the arginine control element that mediates repression and induction by arginine in Saccharomyces cerevisiae
Authors: Crabeel Marjolaine , De Rijcke Martine , Seneca Sara , Heimberg Harry , Pfeiffer Ilona , Matisova Andrea
Published: May 2005

Repression or induction of the genes involved in arginine biosynthesis or catabolism, respectively, both require participation of the ArgRp/Mcm1p regulatory complex. Our previous work showed that those opposite effects were mediated by a similar arginine‐responsive element of 23 nucleotides (that we now call ARC, for ARginine Control) situated close to the start of transcription in the repressed promoters and far upstream of the TATA‐element in the induced promoters.

To define more precisely the sequence and position requirements of the ARC element, we have now characterized by mutagenesis the promoter elements of the arginine‐repressible ARG1 and ARG8 genes. We also identify a functional ARC in the CPA1 promoter, thereby confirming, in agreement with our previous mRNA pulse‐labelling data, the participation of a transcriptional component in the arginine regulation of that gene otherwise submitted to a translational regulation.

From the 12 ARC elements now characterized, we have derived a consensus sequence and show that such a synthetic element is able to mediate ArgRp/Mcm1p‐dependent arginine regulation.

An important new finding illustrated by ARG1 and CPA1, is that contrary to what all the previous data suggested, repression can be mediated by ARC elements located far upstream of the TATA‐box. The new data suggest that the arginine repressor might inhibit transcription in an active process.
Physiological and molecular characterization of flor yeasts: Polymorphism of flor yeast populations
Authors: Martínez P. , Codón A. C. , Pérez L. , Benítez T.
Published: May 2005

Yeast strains which form velum on the surface of Sherry wine during the aging process have been isolated and characterized. According to their metabolic and molecular features most of the yeasts that were isolated belong to different races of Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis and rouxii). Due to the conditions under which these yeasts were isolated, all strains have in common the capacity to develop a film as an adaptive mechanism which allows them to grow and survive in 15·5% vol. ethanol. All strains were prototrophs for amino acids and most vitamins but they gave different responses to the killer factor. However, whereas their physiological features were similar, they showed a great heterogeneity with regards to the nuclear and mitochondrial genome (mtDNA): DNA content per cell was quite variable (1·3 to 2n), electrophoretic karyotypes of nuclear genomes indicated a main pattern with some variations, and polymorphism shown by the mtDNA was very high. Under extreme conditions such as Sherry wine with 15·5% vol. ethanol, no fermentable sugar and an exclusively oxidative metabolism, cells hardly grow and the maintenance of a live population depends on survival and respiration, which in turn depend on the mtDNA. At the same time these environmental conditions are mutagenic for the mtDNA, causing an increase in variation. Thus, the polymorphism observed might reflect the enormous variability induced by the ethanol followed by the selection of those mtDNA sequences which make the mitochondria metabolically active under these conditions.
Carvedilol improves left ventricular function in murine coxsackievirus‐induced acute myocarditis Association with reduced myocardial interleukin‐1β and MMP‐8 expression and a modulated immune response
Authors: Pauschinger Matthias , Rutschow Susanne , Chandrasekharan Kumaran , Westermann Dirk , Weitz Anneke , Schwimmbeck Lothar Peter , Zeichhardt Heinz , Poller Wolfgang , Noutsias Michel , Li Jun , Schultheiss Heinz‐Peter , Tschope Carsten
Published: May 2005

Background

Proinflammatory cytokines induce the expression of matrix metalloproteinases that play a crucial role in myocardial remodeling. Beta‐adrenergic receptor stimulation influences the production of cytokines heralding the possibility of modulating cytokine production by beta‐adrenergic blockers.

Methods and results

In a coxsackievirus B3 murine myocarditis model (BALB/c), effects of carvedilol and metoprolol on myocardial cytokine expression, inflammatory cell infiltration and MMP/TIMP profiles were investigated. In carvedilol‐treated mice, a significant improvement in left ventricular function was documented 10 days post infection. In infected mice (n=10), IL‐1β, TNF‐α, TGF‐β1 and IL‐10 myocardial mRNA abundance were increased significantly (240%, 200%, 161%, and 230%) compared to controls (n=10), while IL‐15 mRNA was markedly reduced (70%). Infected mice showed significantly increased infiltrations with CD3‐, CD4‐ and CD8‐T‐lymphocytes (730%, 1110%, 380%). In the infected mice, myocardial MMP/TIMP profiles presented a significant upregulation of membrane type‐1 MMP, MMP‐9, MMP‐8 and MMP‐3 (150%, 160%, 340%, and 270%) and a significant decrease in TIMP‐4 levels (75%). Carvedilol attenuated over‐expression of myocardial TGF‐β1, IL‐1β and MMP‐8 mRNA expression significantly and induced a relevant IL‐10 mRNA expression in the infected mice (n=10). By an unchanged infiltration with CD3‐T‐lymphocytes, carvedilol showed a representative reduction in CD4‐T‐lymphocytes.

Conclusion

Carvedilol treatment in experimental myocarditis leads to reduced expression of proinflammatory cytokines and MMPs, which contributes to reduced matrix degradation and ultimately to improved structural integrity of the heart. Besides the antiadrenergic potential, carvedilol is beneficial due to a wide range of biological activities (antiinflammatory, antifibrotic, antioxidative and immunomodulatory).
The uptake of D-araboascorbic acid (D-isoascorbic acid) by guinea-pig tissues
Authors: Hughes R. E. , Hurley R. J.
Published: May 2005

1. Guinea-pigs on a low, controlled intake of ascorbic acid (0.3 mg/100 g body-weight) were given daily an oral supplement of L-ascorbic acid (AA) or of D-isoascorbic acid (isoAA) (both 1.5 mg/100 g body-weight). The AA was deposited in the tissues, the isoAA was not.

2. Intramuscularly administered isoAA was deposited in the tissues but not to the same extent as equivalent amounts of intramuscularly administered AA.

3. Paper chromatography confirmed that intramuscularly administered isoAA was deposited in the tissues as isoAA, i.e. there was no conversion into AA.

4. It is concluded that in guinea-pigs (a)AA is absorbed from the gastro-intestinal tract much more readily than isoAA and (b)AA is either more readily abstracted from the blood or is more readily retained by the tissues or both, than is isoAA.
Vitamin E and hepatotoxic agents
Authors: Bunyan J. , Cawthorne M. A. , Diplock A. T. , Green J.
Published: May 2005

1. Fatty liver was induced in 4-month-old male rats by oral dosing with ethanol. The marked increase in liver fat was not accompanied by a rise in lipid peroxides.

2. Homogenates were prepared from the livers of vitamin E-deficient rats and incubated with ethanol. In the concentration range of 10–50 μl/3 ml, ethanol increased the production of malondialdehyde. Methanol, which is not a hepatotoxin, showed a similar effect at 10–35 μl/3 ml homogenate. These findings indicate that the pro-oxidative effect of alcohols in vitro is unrelated to their hepatotoxic action in vivo.

3. Fatty liver was induced in 3.g-month-old, vitamin E-deficient male rats by oral dosingwith ethanol. The effect of pretreatment with vitamin E and N,N'-diphenyl-p-phenylenediamine (DPPD) was studied. D-α-Tocopheryl acetate, given as three doses of 350 mg/kg at 48, 24 and 2 h before the ethanol, failed to decrease the fat accumulation and seemed rather to increase the fat content of the liver. DPPD, given as three doses of 600 mg'kg at similar intervals before the ethanol dose, reduced the fat content of the liver almost to normal.

4. Weanling rats of both sexes were given a vitamin E-deficient diet containing 1% orotic acid for 15 days to induce fatty liver. Dietary supplements of D-a-tocopheryl acetate (500 ppm), selenium (I ppm) or DPPD (100 ppm) did not reduce the lipid accumulation. Lipid peroxides and malondialdehyde levels were lower in the livers of animals treated with orotic acid than in controls, regardless of the presence of vitamin E.

j. Liver necrosis was produced in 9-week-old female vitamin E-deficient rats by the intra-peritoneal injection of zoo mg thioacetamide. Promethazine hydrochloride (Phenergan), given intraperitoneally as two doses (25 mg/kg at the same time as the thioacetamide and 12.5 mg/kg 6 h later), markedly reduced the necrosis. D-α-Tocopheryl acetate, given as two oral doses of 1000mg/kg 48 h and 24 h before the thioacetamide, tended to exacerbate the necrosis.

6. The results are discussed in relation to the question of lipid peroxidation as a cause of hepatotoxicity.
Prediction of carcass fat from body measurements made on live rats differing in age, sex and strain
Authors: Marshall Mary W. , Smith Barbara P. , Munson Arvid W. , Lahmanhn Richard P.
Published: May 2005

1. Individual body fat and body measurements such as lengths, girths and selected skinfold thicknesses were determined in our laboratory strain (BHE), a highly inbred strain (IN) of BHE rats and in a strain of Wistar rats. Measurements were made on unconscious rats in less than 5 min per rat just before autopsy; body fat content was determined in individual rats of both sexes at 50, 100 and 300 days of age.

2. Among the strains, mean total body fat was highest in BHE rats at each age; IN rats of similar average body size were leanest. Statistically significant differences in body fat among the strains were due primarily to differences among the male rats at 300 days. Total protein and skeletal mass increased with increases in age and body size, as did body fat in rats after maturity. IN rats had the largest fat-free weights. Although significant age differences in body fat and body measurements occurred, they were in part dependent upon changes in body- weight, sex and strain of the animals. Female rats had more fat per unit body-weight than males at each age studied. Females, though fatter than males, had smaller skinfold thicknesses, indi- cating that female fat increases are primarily in visceral fat.

3. Large variation in fat among individuals within strains of the same body-weight and age suggests a genetic influence in fat potential in rats not associated with age or body-weight.

4. Results from multiple regression analyses showed differences among adjusted means and partial regression coefficients due to strain and sex of the animals. Although final body-weight was the predictor common to all equations, body length, abdominal girth and subscapular skinfold were common to all but one set of equations. Chest girth, tibia length, triceps and abdominal skinfolds decreased in frequency of appearance in that order. Some of the measurements were more effective predictors at one age than at another. It was clearly necessary to take into account body dimensions other than weight to obtain an optimal prediction of body fat.

5. Differences in characteristics of the rats support the concept of genetic influences in fat deposition in individuals and indicate the complex nature of these influences.
Studies on the oesophageal groove reflex in sheep and on the potential use of the groove to prevent the fermentation of food in the rumen
Authors: Ørskov E. R. , Benzin D.
Published: May 2005

1. Fluids were given by mouth in various ways to young sheep. The extent to which closure of the oesophageal groove operated to direct the fluid to the abomasum was assessed by radiography.

2. Salt solutions and milk given as a drench did not produce complete closure of the groove. However, when the animals were accustomed to sucking from a teat before weaning, any liquid suspension and also water, sucked from a bottle, passed entirely to the abomasum even in 12-month-old sheep.

3. This technique may be useful when it is desirable to cause protein or other nutrients to by-pass fermentation in the rumen.
Metabolism of urea in late pregnancy and the possible contribution of amino acid carbon to glucose synthesis in sheep
Authors: Nolan J. V. , Leng R. A.
Published: May 2005

1. Metabolism of urea in non-pregnant and pregnant sheep (1–25 d from term) has been examined. Injections of [14C]urea were used to estimate urea entry rate, urea pool size and urea space in sheep given 1000 g of a diet of equal parts of crushed oats and chaffed lucerne hay (daya) and in the same sheep 4 d after the ration had been reduced to 250 g (dayb).

2. On both experimental days (aandb), mean pool size was greater (14% on day α, 29% on dayb) and urea space was greater (54% on day α, 24% on dayb) in pregnant animals than in non-pregnant animals; mean plasma urea concentrations were lower (35%) in the pregnant animals on day a but were not significantly different on dayb.

3. The entry rate of urea was similar in all the animals on daya, but was significantly higher (34%) in pregnant than in non-pregnant animals on dayb. There was a significant decrease in urea entry rate in both pregnant (33%) and non-pregnant (86%) animals on dayb.

4. The rate of excretion of urea was lower (26% on daya, 35% on dayb) in pregnant animals, indicating a higher (31% on day α, 40% on dayb) rate of degradation of urea in the digestive tract of pregnant as compared with non-pregnant sheep.

5. Measurements of urea entry rate have been used to calculate the upper limit of amino acid deamination in pregnant and non-pregnant sheep, and this has been used as an indication of the potential availability of amino acid carbon for glucose synthesis. It is suggested that, at a maximum, amino acids may have contributed the carbon required for 63 g/d and 52 g/d of glucose on daysaandbrespectively.
Blood urea measurement as a technique for assessing protein quality
Authors: Eggum Bjørn O.
Published: May 2005

1. Three factors were investigated which influence blood urea content, namely the protein content in the diet, the time after feeding and the protein quality of the diet.

2. To investigate the relationship between the protein content in the diet and the blood urea content, seventeen experiments with rats given increasing amounts of protein in the diet were carried out. The experimental results show that there is a positive correlation (r = 0.95) between the protein content in the diet and the blood urea content.

3. For investigation of the influence of time after feeding on the urea content in the blood, a pig was used having a catheter in the portal vein for blood sampling at intervals. The results from this experiment showed that the blood urea content increases for the first 3–4 h after feeding and thereafter reaches a plateau.

4. To use blood urea measurement as a technique for assessing protein quality it is necessary to work under standardized conditions, especially in regard to the two factors just discussed.

5. Forty-two feeding-stuffs of widely differing quality were used in nitrogen balance trials with rats. The results showed that there is an inverse relation between the blood urea content and the biological value of the diet which is sufficiently accurate (coefficient of variation = 53%) to provide ausefulmethod for the predictionof protein quality from measurement of urea levels.
Evaluation of modified [35S]methionine and [35S]casein preparations as supplements for sheep
Authors: Downes A. M. , Reis P. J. , Sharry L. F. , Tunks D. A.
Published: May 2005

1. Doses of L-[35S]methionine (2 g) and [35S]casein (20 g) were given to sheep in the diet or via the abomasum, and the patterns of 35S-labelling in blood, wool and excreta were studied during the 7 d following administration of the dose.

2. Doses given via the abomasum resulted in substantial labelling of the plasma proteins and wool; only small amounts of the dose were recovered in the urine and faeces. In contrast, doses given in the diet resulted in much less labelling of plasma proteins and wool and ingreater losses in excreta. These results provide the basis of a rapid system for testing the effectiveness of various methods of protecting methionine and casein from degradation in the rumen.

3. From the patterns obtained with N-formyl-DL-[35S]methionine and [35S]polymethionine (mol. wt 7–8 × 104) it appears unlikely that these compounds would stimulate wool growth if given as dietary supplements.

4. [35S]Casein which had been treated with an 8 % aqueous solution of formaldehyde gave results which showed that the casein had been protected from destruction in the rumen without markedly reducing its subsequent digestibility. Treatment of [35S]casein with excess, formalde-hyde (a 40 % aqueous solution) gave a product which was completely indigestible.

5. It is concluded that the radioisotope technique could be applied to other [35S]amino acids or their derivatives and to other [35S]proteins, and should also be applicable to larger ruminants such as cattle.
Effect of protein deficiency on absorption, transport and distribution of α-tocopherol in the rat
Authors: Rajaram O. V. , Fatterpaker Prema , Sreenivasan A.
Published: May 2005

The absorption, transport and distribution of α-[3H]tocopherol were greatly decreased in protein deficiency. This was reflected in the subcellular distribution of α-[3H]tocopherol in livers of protein-deficient rats. The ratio, bound: free for α-[3H]tocopherol, also decreased in both serum and liver cytosol. After protein refeeding, absorption, transport and distribution patterns of α-[3H]tocopherol for the protein-deficient rats were restored to patterns similar to those of control animals.
Nitrogen retention in rats fed on diets enriched with arginine and glycine
Authors: Sitren H. S. , Fisher H.
Published: May 2005

1. Nitrogen retention was measured in adult rats (250–350 g) subjected to the trauma of hind-leg fracture and given diets with or without arginine plus glycine supplementation. Observations were also recorded on excretion of creatine, creatinine, allantoin, and orotic acid. Liver and skeletal muscle transaminase activities were also determined.

2. When traumatized rats weighing approximately 250 g were given a diet with 200 g casein/ kg, supplemented with 20 g arginine and 10 g glycine/kg (EC diet) or a casein diet made isonitrogenous with the EC diet by addition of aspartic acid (C diet), a 60–70% increase in N retention was observed for the first 5 d post-injury for animals consuming the EC diet. A soyabean (S) diet, isonitrogenous to the diet containing 20% casein, supplemented with arginine and glycine was as effective as the EC diet in promoting significantly better N retention of traumatized rats (350 g) in comparison to rats given the C diet.

3. When the dietary casein content was reduced to 100 g/kg, supplements of 10 g arginine and 5 g glycine or 20 g arginine and 10 g glycine/kg did not improve N retention. It is suggested that both protein quality and protein quantity are important following injury.

4. An increased excretion of creatine was observed in traumatized rats given the high-protein diets supplemented with arginine and glycine. No consistent changes were noted for urine creatinine.

5. Urine allantoin levels remained stable after leg-fracture in rats consuming either the C or EC diets. Differences in the levels of urine orotic acid were found during both the pre- and post-injury periods in rats given the C, EC or S diets.

6. The mechanisms responsible for the improved N retention of traumatized rats consuming the high-protein diets with supplements of arginine and glycine may be related to the role of arginine both as a constituent of muscle tissue and as an intermediate in the urea cycle.

7. In traumatized rats fed the C or EC diets, liver transaminase activity increased whereas the transaminase activity in skeletal muscle decreased. These results support the recent concept that the increased excretion of N following injury arises from diminished reutilization of amino acids by muscle tissue without an acute increase in the rate of muscle catabolism.
Major Signaling Pathways Modulate Arabidopsis Glucosinolate Accumulation and Response to Both Phloem-Feeding and Chewing Insects
Authors: Mewis Inga , Appel Heidi M. , Hom Amanda , Raina Ramesh , Schultz Jack C.
Published: May 2005

Plant responses to enemies are coordinated by several interacting signaling systems. Molecular and genetic studies with mutants and exogenous signal application suggest that jasmonate (JA)-, salicylate (SA)-, and ethylene (ET)-mediated pathways modulate expression of portions of the defense phenotype in Arabidopsis (Arabidopsis thaliana), but have not yet linked these observations directly with plant responses to insect attack. We compared the glucosinolate (GS) profiles of rosette leaves of 4-week-old mutant and transgenic Arabidopsis (Columbia) plants compromised in these three major signaling pathways, and characterized responses by those plants to feeding by two phloem-feeding aphids (generalist Myzus persicae and specialist Brevicoryne brassicae) and one generalist caterpillar species (Spodoptera exigua Hubner). Blocked JA signaling in coronatine-insensitive (coi1) and enhanced expression of SA-signaled disease resistance in hypersensitive response-like (hrl1) mutants reduced constitutive GS concentrations, while blocking SA signaling at the mediator protein npr1 mutant (NPR) increased them. There was no significant impact on constitutive GS contents of blocking ET signaling (at ET resistant [etr1]) or reducing SA concentrations (nahG transgene). We found increased GS accumulation in response to insect feeding, which required functional NPR1 and ETR1 but not COI1 or SA. Insect feeding caused increases primarily in short-chain aliphatic methylsulfinyl GS. By contrast, responses to exogenous JA, a frequent experimental surrogate for insect attack, were characterized by an increase in indolyl GS. Insect performance, measured as population increase or weight increase, was negatively related to GS levels, but we found evidence that other, ET-regulated factors may also be influential. Plant resistance to (consumption by) S. exigua was not related to insect growth because some plant chemistries inhibited growth while others inhibited feeding. These major signaling pathways modulate Arabidopsis GS accumulation and response to both phloem-feeding and chewing insects, often antagonistically; NPR appears to be central to these interactions. Our results indicate that exogenous signal application and plant consumption measures may not provide useful measures of plant responses to actual insect feeding.
Structural Complexity, Differential Response to Infection, and Tissue Specificity of Indolic and Phenylpropanoid Secondary Metabolism in Arabidopsis Roots
Authors: Bednarek Paweł , Schneider Bernd , Svatoš Aleš , Oldham Neil J. , Hahlbrock Klaus
Published: May 2005

Levels of indolic and phenylpropanoid secondary metabolites in Arabidopsis (Arabidopsis thaliana) leaves undergo rapid and drastic changes during pathogen defense, yet little is known about this process in roots. Using Arabidopsis wild-type and mutant root cultures as an experimental system, and the root-pathogenic oomycete, Pythium sylvaticum, for infections, we analyzed the aromatic metabolite profiles in soluble extracts from uninfected and infected roots, as well as from the surrounding medium. A total of 16 indolic, one heterocyclic, and three phenylpropanoid compounds were structurally identified by mass spectrometry and nuclear magnetic resonance analyses. Most of the indolics increased strongly upon infection, whereas the three phenylpropanoids decreased. Concomitant increases in both indolic and phenylpropanoid biosynthetic mRNAs suggested that phenylpropanoids other than those examined here in “soluble extracts” were coinduced with the indolics. These and previous results indicate that roots differ greatly from leaves with regard to the nature and relative abundance of all major soluble phenylpropanoid constituents. For indolics, by contrast, our data reveal far-reaching similarities between roots and leaves and, beyond this comparative aspect, provide an insight into this highly diversified yet under-explored metabolic realm. The data point to metabolic interconnections among the compounds identified and suggest a partial revision of the previously proposed camalexin pathway.
The AtRAD51C Gene Is Required for Normal Meiotic Chromosome Synapsis and Double-Stranded Break Repair in Arabidopsis
Authors: Li Wuxing , Yang Xiaohui , Lin Zhenguo , Timofejeva Ljudmilla , Xiao Rong , Makaroff Christopher A. , Ma Hong
Published: May 2005

Meiotic prophase I is a complex process involving homologous chromosome (homolog) pairing, synapsis, and recombination. The budding yeast (Saccharomyces cerevisiae) RAD51 gene is known to be important for recombination and DNA repair in the mitotic cell cycle. In addition, RAD51 is required for meiosis and its Arabidopsis (Arabidopsis thaliana) ortholog is important for normal meiotic homolog pairing, synapsis, and repair of double-stranded breaks. In vertebrate cell cultures, the RAD51 paralog RAD51C is also important for mitotic homologous recombination and maintenance of genome integrity. However, the function of RAD51C in meiosis is not well understood. Here we describe the identification and analysis of a mutation in the Arabidopsis RAD51C ortholog, AtRAD51C. Although the atrad51c-1 mutant has normal vegetative and flower development and has no detectable abnormality in mitosis, it is completely male and female sterile. During early meiosis, homologous chromosomes in atrad51c-1 fail to undergo synapsis and become severely fragmented. In addition, analysis of the atrad51c-1 atspo11-1 double mutant showed that fragmentation was nearly completely suppressed by the atspo11-1 mutation, indicating that the fragmentation largely represents a defect in processing double-stranded breaks generated by AtSPO11-1. Fluorescence in situ hybridization experiments suggest that homolog juxtaposition might also be abnormal in atrad51c-1 meiocytes. These results demonstrate that AtRAD51C is essential for normal meiosis and is probably required for homologous synapsis.
Arabidopsis Peroxisomal Citrate Synthase Is Required for Fatty Acid Respiration and Seed Germination
Authors: Pracharoenwattana Itsara , Cornah Johanna E. , Smith Steven M.
Published: May 2005

We tested the hypothesis that peroxisomal citrate synthase (CSY) is required for carbon transfer from peroxisomes to mitochondria during respiration of triacylglycerol in Arabidopsis thaliana seedlings. Two genes encoding peroxisomal CSY are expressed in Arabidopsis seedlings, and seeds from plants with both CSY genes disrupted were dormant and did not metabolize triacylglycerol. Germination was achieved by removing the seed coat and supplying sucrose, but the seedlings still did not use triacylglycerol. The mutant seedlings were resistant to 2,4-dichlorophenoxybutyric acid, indicating a block in peroxisomal β-oxidation, and were unable to develop further after transfer to soil. The mutant phenotype was complemented with a cDNA encoding CSY with either its native peroxisomal targeting sequence (PTS2) or a heterologous PTS1 sequence from pumpkin (Cucurbita pepo) malate synthase. These results suggest that peroxisomal CSY in Arabidopsis is not only a key enzyme of the glyoxylate cycle but also catalyzes an essential step in the respiration of fatty acids. We conclude that citrate is exported from the peroxisome during fatty acid respiration, whereas in yeast, acetylcarnitine is exported.
Hepatic venoconstriction is involved in anaphylactic hypotension in rats
Authors: Shibamoto Toshishige , Cui Sen , Ruan Zonghai , Liu Wei , Takano Hiromichi , Kurata Yasutaka
Published: May 2005

We determined the roles of liver and splanchnic vascular bed in anaphylactic hypotension in anesthetized rats and the effects of anaphylaxis on hepatic vascular resistances and liver weight in isolated perfused rat livers. In anesthetized rats sensitized with ovalbumin (1 mg), an intravenous injection of 0.6 mg ovalbumin caused not only a decrease in systemic arterial pressure from 120 ± 9 to 43 ± 10 mmHg but also an increase in portal venous pressure that persisted for 20 min after the antigen injection (the portal hypertension phase). The elimination of the splanchnic vascular beds, by the occlusions of the celiac and mesenteric arteries, combined with total hepatectomy attenuated anaphylactic hypotension during the portal hypertension phase. For the isolated perfused rat liver experiment, the livers derived from sensitized rats were hemoperfused via the portal vein at a constant flow. Using the double-occlusion technique to estimate the hepatic sinusoidal pressure, presinusoidal ( Rpre) and postsinusoidal ( Rpost) resistances were calculated. An injection of antigen (0.015 mg) caused venoconstriction characterized by an almost selective increase in Rpre rather than Rpost and liver weight loss. Taken together, these results suggest that liver and splanchnic vascular beds are involved in anaphylactic hypotension presumably because of anaphylactic presinusoidal contraction-induced portal hypertension, which induced splanchnic congestion resulting in a decrease in circulating blood volume and thus systemic arterial hypotension.
The changing role of eosinophils in long-term hyperreactivity following a single ozone exposure
Authors: Yost Bethany L. , Gleich Gerald J. , Jacoby David B. , Fryer Allison D.
Published: May 2005

Ozone hyperreactivity over 24 h is mediated by blockade of inhibitory M2muscarinic autoreceptors by eosinophil major basic protein. Because eosinophil populations in the lungs fluctuate following ozone, the contribution of eosinophils to M2dysfunction and airway hyperreactivity was measured over several days. After one exposure to ozone, M2function, vagal reactivity, smooth muscle responsiveness, and inflammation were measured in anesthetized guinea pigs. Ozone-induced hyperreactivity to vagal stimulation persisted over 3 days. Although hyperreactivity one day after ozone is mediated by eosinophils, AbVLA-4 did not inhibit either eosinophil accumulation in the lungs or around the nerves or prevent hyperreactivity at this time point. Two days after ozone, eosinophils in BAL, around airway nerves and in lungs, were decreased, and neuronal M2receptor function was normal, although animals were still hyperreactive to vagal stimulation. Depleting eosinophils with AbIL-5 prevented hyperreactivity, thus eosinophils contribute to vagal hyperreactivity by mechanisms separate from M2receptor blockade. Three days after ozone, vagal hyperreactivity persisted, eosinophils were again elevated in BAL in lungs and around nerves, and M2receptors were again dysfunctional. At this point, airway smooth muscle was also hyperresponsive to methacholine. Eosinophil depletion with AbIL-5, AbVLA-4, or cyclophosphamide protected M2function 3 days after ozone and prevented smooth muscle hyperreactivity. However, vagal hyperreactivity was significantly potentiated by eosinophil depletion. The site of hyperreactivity, muscle or nerve, changes over 3 days after a single exposure to ozone. Additionally, the role of eosinophils is complex; they mediate hyperreactivity acutely while chronically may be involved in repair.