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M Rose

Publications and source records attributed to M Rose.

At least 19 recordsLinked to original sources
PubMed1995

Sequence and functional analysis of a 7.2 kb fragment of Saccharomyces cerevisiae chromosome II including GAL7 and GAL10 and a new essential open reading frame.

The nucleotide sequence of a fragment of 7200 base pairs of Saccharomyces cerevisiae chromosome II has been determined. The sequence contains three open reading frames (ORFs). Two genes for galactose metabolism, GAL7 and part of the GAL10 coding region, are localized on the fragment. Comparison to the previously published sequence data showed several differences, leading to changes in the amino acid sequences of GAL7 and GAL10. One new ORF, YBR0224, was detected, coding for a protein with 918 amino acids. Comparison to the DNA and protein data bases showed no significant homologies. The protein has some interesting features pointing to a function involved in transcription regulation: a leucine zipper motif, a highly acidic region, possibly involved in transcription activation and a putative nuclear localization signal. Deletion analysis showed that the gene is essential when deleted in strain W303. Spores could germinate and form microcolonies, but efforts to propagate the colonies failed. Deletion of this gene in a different genetic background (strain M5) led to very poor-growing mutant strains with cells showing aberrant cellular morphologies.

Amino Acid Sequence↗
PubMed1995

Sequence and functional analysis of a 7.2 kb DNA fragment containing four open reading frames located between RPB5 and CDC28 on the right arm of chromosome II.

In a coordinated approach, several laboratories sequenced Saccharomyces cerevisiae chromosome II during the European BRIDGE project. Here we report on the sequence and functional analysis of a 7217 bp fragment located on the right arm of chromosome II between RPB5 and CDC28. The fragment contains four open reading frames probably encoding proteins of 79.2 kDa (corresponding gene YBR156c), 12.1 kDa (YBR157c), 62.7 kDa (YBR158w) and 38.7 kDa (YBR159w). All four open reading frames encode new proteins, as concluded from data base searches. The respective genes were destroyed by gene replacement in one allele of diploid cells. After sporulation and tetrad analysis, the resulting mutant haploid strains were investigated. No phenotype with respect to spore germination, viability, carbohydrate utilization, and growth was found for YBR157c, encoding the smallest open reading frame investigated. Gene replacement within the YBR156c gene encoding a highly basic and possibly nuclear located protein was lethal. Ybr158 revealed similarities to the Grrl (Cat80) protein with respect to the leucine-rich region. Cells harboring a mutation in the YBR158w gene showed strongly reduced growth as compared to the wild-type cells. The protein predicted from YBR159w shared 33% identical amino acid residues with the human estradiol 17-beta-hydroxysterol dehydrogenase 3. Haploid ybr159c mutants were only able to grow at reduced temperatures, but even under these conditions the mutants grew slower than wild-type strains.

Amino Acid Sequence↗
PubMed1995

Molecular analysis of the yeast SER1 gene encoding 3-phosphoserine aminotransferase: regulation by general control and serine repression.

Although serine and glycine are ubiquitous amino acids the genetic and biochemical regulation of their synthesis has not been studied in detail. The SER1 gene encodes 3-phosphoserine aminotransferase which catalyzes the formation of phosphoserine from 3-phosphohydroxy-pyruvate, which is obtained by oxidation of 3-phosphoglycerate, an intermediate of glycolysis. Saccharomyces cerevisiae cells provided with fermentable carbon sources mainly use this pathway (glycolytic pathway) to synthesize serine and glycine. We report the isolation of the SER1 gene by complementation and the disruption of the chromosomal locus. Sequence analysis revealed an open reading frame encoding a protein with a predicted molecular weight of 43,401 Da. A previously described mammalian progesterone-induced protein shares 47% similarity with SER1 over the entire protein, indicating a common function for both proteins. We demonstrate that SER1 transcription is regulated by the general control of amino-acid biosynthesis mediated by GCN4. Additionally, DNaseI protection experiments proved the binding of GCN4 protein to the SER1 promoter in vitro and three GCN4 recognition elements (GCREs) were identified. Furthermore, there is evidence for an additional regulation by serine end product repression.

Amino Acid Sequence↗
PubMed1995

Molecular and biochemical characterization of the hexokinase from the starch-utilizing yeast Schwanniomyces occidentalis.

Hexose-phosphorylating enzymes from the starch-utilizing yeast Schwanniomyces occidentalis were purified and two isoenzymes separated. The substrate pattern characterized one of these as a hexokinase phosphorylating glucose and fructose and the other as a glucokinase unable to phosphorylate fructose. The purified Schw. occidentalis hexokinase had a KM value of 0.98 mM for glucose and 9.3 mM for fructose. The hexokinase gene was cloned by cross hybridization with a probe from the Saccharomyces cerevisiae HXK2 gene. Deletion of Schw. occidentalis hexokinase by gene replacement yielded a mutant unable to grow on fructose as sole carbon source, but still growing on glucose. Deletion mutants of Schw. occidentalis hexokinase prevented glucose repression of invertase and maltase. Growth deficiencies and the defect of glucose repression of a S. cerevisiae hexokinase null mutant could be restored by heterologous expression of the Schw. occidentalis hexokinase. Moreover, the results clearly showed the existence of a separate glucokinase in Schw. occidentalis.

Amino Acid Sequence↗
PubMed1995

A pharmacokinetic comparison of cyclosporin oral solution and cyclosporin capsules in heart and lung transplant recipients.

Pharmacokinetic profiles were obtained for 16 heart or lung recipients following the administration of identical doses of cyclosporin as oral solution and capsules on consecutive days. A comparison of pharmacokinetic parameters (AUC, Cmax, Cmin and tmax) showed that there were no significant differences between the two formulations except for the tmax, which was significantly longer for the capsules. The mean variation in day-to-day trough levels produced by the two different forms was 25.6%. A retrospective study was carried out of consecutive cyclosporin levels in patients at steady state on oral solution. The mean variation in day-to-day trough levels was 32.3%. This was not significantly different from the variation in consecutive trough levels seen in the oral solution/capsule comparison. This study shows that cyclosporin capsules can be substituted for oral solution without causing acute changes in cyclosporin blood levels, and that the pharmacokinetics of the two formulations are similar.

Administration, Oral↗
PubMed1995

Characterization of a rat anterior pituitary cell bioassay.

We have described the protocols and characterization of a pituicyte culture, which became established as a reliable and reproducible bioassay for the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The bioassay was used to measure the bioactivity of factors that inhibit and stimulate gonadotrophin secretion. The protocol that was used involved the culling of female Wistar rats (200 to 250 g weight), at random stages of their cycle, and dispersal of their pituicytes in a concentration of 0.4 x 10(6) cells.ml-1.well-1 in serum-free medium (Dulbecco's modified Eagle's medium/Ham's F12 mixture, supplemented with insulin and transferrin) in Falcon 3047 24-well culture plates. After 24 h of pre-culture, the medium was changed and the cells cultured for a further 48 h. The supernatant was removed and assayed for basal secretion of FSH and LH. The cells were then stimulated with 10(-8) M GnRH for 4 h and the supernatant assayed for gonadotrophin-releasing hormone (GnRH)-stimulated FSH and LH secretion. All samples were assayed as pairs of duplicates (i.e. quadruplicate samples) which were randomly added to the plates to minimize plate effects. Random number tables were used to achieve this randomization.

Animals↗
PubMed1995

Immunomodulation by theophylline in asthma. Demonstration by withdrawal of therapy.

Theophylline is the most widely used anti-asthma drug worldwide and is classified as a bronchodilator, although there is increasing evidence that it may have immunomodulatory effects. We have investigated the effects of theophylline withdrawal under placebo control in 27 asthmatic patients (25 to 70 yr) treated with long-term theophylline who were also treated with high dose inhaled corticosteroids. We measured asthma symptoms (diary card), lung function (spirometry and home records of peak expiratory flow), and peripheral leukocyte populations using dual color flow cytometry. In eight of these patients, we examined fiberoptic bronchial biopsies by immunocytochemistry. We also studied peripheral blood lymphocytes in eight asthmatic patients who have never received theophylline. Mean steady state plasma theophylline concentrations during theophylline therapy were 8.6 +/- 0.9 mg/L. Theophylline withdrawal was associated with a significant increase in asthma symptoms, particularly at night, and a fall in spirometry and morning peak flow. This was accompanied by a significant fall in peripheral blood monocytes (CD14+, activated CD4+ T-lymphocytes (CD4+/CD25+) and activated CD8+ T-cells (CD8+/HLA-DR+) in patients with a plasma theophylline > 5 mg/L. The lymphocyte populations in theophylline-naive patients were similar to those found after theophylline withdrawal. Bronchial biopsies showed a mirror image of the peripheral blood with an increase in CD4+ and CD8+ lymphocytes in the airway. Chronic treatment with theophylline, even at low plasma concentrations, controls asthma symptoms and has effects on T-lymphocyte populations in the peripheral blood which are the inverse of those observed in the airways.(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗
PubMed1995

In vitro bioactivity of gonadotrophin surge attenuating factor is not affected by an antibody to human inhibin.

The effect of anti-inhibin antibodies on gonadotrophin surge attenuating/inhibiting factor (GnSAF/GnSIF) and its effect on gonadotrophin secretion in a pituitary cell bioassay were determined by culturing rat pituitary cells in a serum-free medium to which inhibin and a partially purified preparation of gonadotrophin surge inhibiting factor (GnSIF) were added. The samples were treated with an anti-inhibin antiserum and basal and GnRH-stimulated FSH and LH secretion were measured by radioimmunoassay of the culture supernatant. Inhibin had a dose-dependent inhibitory effect on both basal and GnRH-stimulated FSH secretion and also on GnRH-stimulated LH secretion. Anti-inhibin antibody blocked the inhibition of FSH secretion, except at the highest two doses of human inhibin (1.25 ng ml-1, P = 0.04; 2.5 ng ml-1, P = 0.0004). The GnSIF preparation inhibited basal and GnRH-stimulated FSH and GnRH-stimulated LH secretion and was not affected by the anti-inhibin antibody. The novel hormone, GnSIF, is different from inhibin.

Animals↗
PubMed1995

Differences in carbohydrate composition of FSH preparations detected with lectin-ELISA systems.

FSH is a glycoprotein containing N-linked carbohydrates which exhibit a variety of forms ranging from mono- to multibranched structures. Variation in glycosylation, particularly the degree of terminal sialylation, determines the half-life of the hormone and hence its in vivo bioactivity. The glycoform content of FSH preparations can differ according to the source (e.g. pituitary, urine), cell line (for rDNA-derived material) and selectivity of purification procedures, and may create difficulties in the preparation and characterization of standards and therapeutic products. In order to develop a simple method to detect changes in glycocomposition, an FSH ELISA was modified by the incorporation of lectins of recognized sugar specificity, and used to examine the terminal sugar composition of ampouled preparations of pituitary, urinary and rDNA-derived FSH. FSH was captured with a specific monoclonal antibody (MAb) and detected with either biotinylated anti-FSH MAb (ELISA) or the sugar-specific lectins (L-ELISA) from Triticum vulgaris (sialic acid, SA), Sambucus nigra (alpha 2,6-linked SA), Maackia amurensis (alpha 2,3-linked SA) or Ricinus communis (free terminal galactose; GAL). Relative estimates of the amounts of terminal SA, its different forms and GAL were derived from the L-ELISA/ELISA data compared with the highly sialylated 1st International Standard for pituitary FSH (IS) 83/575. All the FSH preparations had less SA than the IS with the ratio of alpha 2,3- and alpha 2,6-linked SA varying between preparations. The amounts of alpha 2,6-linked SA relative to the IS were not significantly different in the urinary and pituitary preparations whereas alpha 2,3-linked SA in all preparations was generally less than that of the standard.(ABSTRACT TRUNCATED AT 250 WORDS)

Carbohydrate Conformation↗
PubMed1995

[The immune response in edema disease of weaned piglets measured with a recombinant B subunit of shiga-like toxin II].

An outbreak of edema disease (ED) was monitored in 80 piglets after weaning over a period of 4 weeks. The shedding of Shiga-like toxin-IIe) producing Escherichia coli strains, the serum bactericidal activity (SBA) against SLTEC-IIe, and the antibody response against SLT-IIe were investigated. The antibody response was monitored by utilizing a glutathione-S-transferase (GST) + SLT-IIe B/SUB fusion protein (FRANKE et al., in press) for immunoblot assays. E. coli-strain GO15III (0141:K85ac) was diagnosed as SLT-IIe-producing E. coli by polymerase chain reaction, DNA hybridization and cytotoxicity assays. Maximum excretion of GO15III appeared between days 8 and 15 after weaning. On day 1 after weaning no piglet shed GO15III, while the number increased on day 8 to 53 (66.2%) and on day 15 to 59 (73.8%) of the piglets. 4 week after weaning, GO15III was only isolated from 23 (28.8%) of the piglets. In parallel, serum bactericidal activity against GO15III increased significantly in the sera of 73 (91.2%) piglets, reaching a stable maximum from day 15 on. During the first two weeks after weaning, no piglet yielded detectable SLT-IIe-IgG. However, the number of SLT-IIe-IgG positive piglets increased steadily from day 15. On day 15, 5 (6.2%) piglets were positive in SLT-IIe immunoblot analysis and 29 days after weaning the number increased to 31 (38.8%). These data represent the first serological monitoring of a natural outbreak of edema disease in piglets after weaning by using a recombinant fusion protein (GST+SLT-IIe B/SUB). The recombinant protein proved to be a useful diagnostical tool for monitoring the specific antibody status of piglets.

Animals↗
PubMed1994

Complete DNA sequence of yeast chromosome II.

In the framework of the EU genome-sequencing programmes, the complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome II (807 188 bp) has been determined. At present, this is the largest eukaryotic chromosome entirely sequenced. A total of 410 open reading frames (ORFs) were identified, covering 72% of the sequence. Similarity searches revealed that 124 ORFs (30%) correspond to genes of known function, 51 ORFs (12.5%) appear to be homologues of genes whose functions are known, 52 others (12.5%) have homologues the functions of which are not well defined and another 33 of the novel putative genes (8%) exhibit a degree of similarity which is insufficient to confidently assign function. Of the genes on chromosome II, 37-45% are thus of unpredicted function. Among the novel putative genes, we found several that are related to genes that perform differentiated functions in multicellular organisms of are involved in malignancy. In addition to a compact arrangement of potential protein coding sequences, the analysis of this chromosome confirmed general chromosome patterns but also revealed particular novel features of chromosomal organization. Alternating regional variations in average base composition correlate with variations in local gene density along chromosome II, as observed in chromosomes XI and III. We propose that functional ARS elements are preferably located in the AT-rich regions that have a spacing of approximately 110 kb. Similarly, the 13 tRNA genes and the three Ty elements of chromosome II are found in AT-rich regions. In chromosome II, the distribution of coding sequences between the two strands is biased, with a ratio of 1.3:1. An interesting aspect regarding the evolution of the eukaryotic genome is the finding that chromosome II has a high degree of internal genetic redundancy, amounting to 16% of the coding capacity.

Base Composition↗
PubMed1994

Comparative studies of UV-induced DNA cleavage by structural isomers of an iodinated DNA ligand.

PURPOSE: To evaluate the importance of the position of the halogen atom in iodinated DNA-binding bibenzimidazoles, with respect to sensitization of UV-A-induced DNA breakage. METHODS AND MATERIALS: Three analogues of iodoHoechst 33258, denoted ortho-, meta- and paraiodoHoechst, according to the site of iodine substitution, were synthesized. Plasmid DNA (pBR322) was used to assay UV-A-induced DNA single-strand breaks (ssbs). The location of the sites of strand breakage was determined by DNA sequencing gel analysis, using a 32P-endlabelled oligoDNA with a single binding site for the ligands. RESULTS: A clear trend in decreasing activity of sensitization of UV-induced DNA ssbs was established: ortho- > meta-, para- > iodoHoechst 33258. The sequencing gel studies showed that orthoiodoHoechst was distinct from the other three compounds, with respect to the sites of DNA strand breakage and the chemistry of the cleavage reaction. CONCLUSION: The position of iodine substitution in iodinated bibenzimidazoles determines the location of the carbon-centered radical on the ligand in the minor groove of DNA. DNA strand cleavage is mediated by abstraction of a nearby deoxyribosyl H-atom. Hence, the position of the radical species determines: which deoxyribosyl group is attacked (i.e., site of cleavage relative to the ligand binding site); which H-atom is abstracted, more specifically which of the five deoxyribosyl carbons is involved (i.e., the chemistry of the cleavage reaction), and the stereochemistry of the transition state for the H-atom abstraction (and hence the efficiency or extent of strand breakage). The ortho-compound represents the best example to date of iodinated DNA ligands designed as potential radiation sensitizers, as an extension of the well-established sensitization by halogenated DNA precursors.

Base Sequence↗
PubMed1994

Human endothelial stimulation of allogeneic T cells via a CTLA-4 independent pathway.

It is accepted that T cells require at least two signals to undergo proliferation and cytokine release: an antigen dependent signal mediated via the the TCR (T cell receptor) and an antigen independent signal mediated via one or more accessory or adhesion molecules. Interaction between CD28 or CTLA-4 and the B7 co-receptors found on many antigen presenting cells (APC) is known to be essential for antigen specific (including alloantigen) expansion of T cells in vitro and in vivo. CTLA-4-Ig is a fusion protein with very high affinity for B7. It has been used in vivo to block both allograft and xenograft rejection. Most of the work investigating second signal requirement has used 'professional' APC. In view of the observations that class II positive human endothelial cells can cause direct allostimulation of resting CD4+ and CD8+ T cells, we have investigated the requirement of CTLA-4 in this response. The current studies show that the proliferative response of allogeneic CD4+ and CD8+ T cells to interferon-gamma treated HUVEC (human umbilical vein endothelial cells) is inhibited by monoclonal antibodies (mAbs) against MHC class II and class I antigens, respectively, but not by CTLA-4-Ig. In contrast, lymphocytes proliferating in response to allogeneic splenocytes are inhibited by CTLA-4-Ig. Cell surface binding studies using flow cytometry demonstrated failure of endothelial cells to bind either CTLA-4-Ig or mAbs against B7 receptors. In conclusion, different APC use different co-stimulatory signals. The possibility that this leads to different cytokine profiles needs to be investigated to further understand the role of endothelial cells in transplant rejection.

Abatacept↗
PubMed1994

The prevention of spread of methicillin resistant Staphylococcus aureus in a spinal injuries centre.

The National Spinal Injuries Centre (NSIC) is a tertiary referral centre. It accepts most of its patients from other hospitals in the UK and overseas. The severity of injury, the presence of a tracheostomy, urinary catheter and pressure sores predisposes this group of patients to colonisation or infection with Methicillin resistant Staphylococcus aureus (MRSA). The NSIC uses simple but strict protocols for hygiene, screening for MRSA, and source isolation of known or suspected MRSA carriers in single room accommodation to control the spread of MRSA in the centre. A retrospective search of microbiology and patient records revealed that in 4 years there had been 24 admissions with MRSA, with a total of 1421 isolation days. There was only one outbreak of MRSA. This involved three patients. Hygiene, screening of potential MRSA carriers together with single room isolation can limit the spread of MRSA.

Cross Infection↗
PubMed1994

Comparative studies of UV-induced DNA cleavage by analogues of iodoHoechst 33258.

Following the earlier demonstration that iodo-Hoechst 33258 sensitizes DNA and cells to UVA, presumably mediated by formation of a carbon-centred radical on the ligand upon dehalogenation, three isomeric analogues of iodo-Hoechst 33258 have now been studied. The isomers differ in the location of the iodine atom in the phenyl ring of the ligand, relative to the site of attachment of the bibenzimidazole moiety, and are accordingly denoted ortho-, meta- and para-iodoHoechst. Comparison of the ligands with respect to induction of DNA ssb in pBR322 DNA revealed a wide range of activity; (D37's vary by a factor of 37), decreasing in the order: ortho- > meta- and para- > iodoHoechst 33258. Preliminary dehalogenation studies suggest that the higher activity of the ortho isomer results more from increased cross-section for dehalogenation than from increased efficiency of strand breakage per dehalogenation event. However, the chemistry of strand breakage by the ortho-isomer is distinctive, and tentatively assigned to initial attack at the 1'-deoxyribosyl carbon; the other two isomers, like iodo-Hoechst 33258, attack the 5'-carbon. The results are discussed in terms of the spectrum of DNA strand breakage chemistry associated with ionizing radiation, and the potential of DNA strand breaking agents such as the iodoHoechst compounds to study the chemical and biological consequences of the different subclasses of initial DNA damage.

Base Sequence↗
PubMed1994

The combined use of soybean agglutinin and immunomagnetic beads for T lymphocyte subset depletion of bone marrow allografts: a laboratory analysis.

The prevention of graft-versus-host disease by T-lymphocyte depletion of allografts prior to bone marrow transplantation has resulted in an increase in graft failure/rejection and relapse of disease. Evidence for the selective roles of specific T-lymphocyte subsets in each of the engraftment, graft-versus-host disease, and disease relapse processes has been presented, but sufficient clinical verification to support any hypothesis in this regard is lacking. In this paper we describe a convenient and highly flexible clinical laboratory method for depletion of specific T-lymphocytes in controllable quantities by the use of select monoclonal antibodies. Almost 3 log10 removal of CD2+ and CD8+ cells without significant loss of hematopoietic progenitors (CFU-GM, BFU-E, and CD34+ cells) can be reproducibly achieved. This method, employing soybean agglutination and immunomagnetic beads, is potentially adaptable to depletion of any cell subset or any tumor cell for which unique cell-surface antigen characteristics have been defined. In addition, our protocol is equally suited to the positive selection of stem cells and hematopoietic progenitors bearing the CD34 surface antigen.

Bone Marrow Transplantation↗