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Biomedical subjects

A D Johnson

Publications and source records attributed to A D Johnson.

At least 19 recordsLinked to original sources
PubMed1992

Ssn6-Tup1 is a general repressor of transcription in yeast.

The homeodomain protein alpha 2 and the SRF-like protein Mcm1 are required to establish cell type in the yeast Saccharomyces cerevisiae. Together, these regulatory proteins recognize a specific DNA operator, marking a set of genes for transcriptional repression. In this paper, we show that occupancy of the operator by alpha 2-Mcm1 is not sufficient to bring about repression. Rather, repression is effected only when Ssn6 (a TPR protein) and Tup1 (a beta-transducin repeat protein) are also present in the cell. We show that Ssn6 represses transcription when brought to a promoter by a bacterial DNA-binding domain and that Tup1 is required for this repression. Based on these and other results, we propose that Ssn6-Tup1 is a general repressor of transcription in yeast, recruited to target promoters by a variety of sequence-specific DNA-binding proteins.

Immunoblotting↗
PubMed1992

A molecular mechanism for combinatorial control in yeast: MCM1 protein sets the spacing and orientation of the homeodomains of an alpha 2 dimer.

DNA recognition sequences for dimeric proteins typically contain two types of information. The first is the DNA sequence of each half-site, and the second is the arrangement of these half-sites. We show that dimers of the yeast homeodomain protein alpha 2, although able to read the first type of information, lack the ability to assess the second type. Rather, alpha 2 dimers bind with equal affinity to artificial operators in which the two half-sites are arrayed as inverted repeats, as direct repeats, or as everted (inside-out) repeats. We show that a second protein-MCM1-sets the exact spacing and orientation of the homeodomains in the alpha 2 dimer so that they accommodate only the geometry of the naturally occurring operators. These experiments show directly how the target specificity of a homeodomain protein is raised by an auxiliary protein, allowing it to distinguish the biologically correct operators from closely related sequences in the cell.

Base Sequence↗
PubMed1992

Meiotic induction of the yeast HOP1 gene is controlled by positive and negative regulatory sites.

The process of meiosis and sporulation in the yeast Saccharomyces cerevisiae is a highly regulated developmental pathway dependent on genetic as well as nutritional signals. The HOP1 gene, which encodes a component of meiotic chromosomes, is not expressed in mitotically growing cells, but its transcription is induced shortly after yeast cells enter the meiotic pathway. Through a series of deletions and mutations in the HOP1 promoter, we located two regulatory sites that are essential for proper regulation of HOP1. One site, called URS1H, brings about repression of HOP1 in mitotic cells and functions as an activator sequence in cells undergoing meiosis. The second site, which we designated UASH, acts as an activator sequence in meiotic cells and has similarity to the binding site of the mammalian CCAAT/enhancer binding protein (C/EBP). Both sites are required for full meiotic induction of the HOP1 promoter. We conclude that in mitotic yeast cells, the URS1H site maintains the repressed state of the HOP1 promoter, masking the effect of the UASH site. Upon entry into meiosis, repression is lifted, allowing the URS1H and UASH sites to activate high-level transcription.

Base Sequence↗
PubMed1991

Crystal structure of a MAT alpha 2 homeodomain-operator complex suggests a general model for homeodomain-DNA interactions.

The MAT alpha 2 homeodomain regulates the expression of cell type-specific genes in yeast. We have determined the 2.7 A resolution crystal structure of the alpha 2 homeodomain bound to a biologically relevant DNA sequence. The DNA in this complex is contacted primarily by the third of three alpha-helices, with additional contacts coming from an N-terminal arm. Comparison of the yeast alpha 2 and the Drosophila engrailed homeodomain-DNA complexes shows that the protein fold is highly conserved, despite a 3-residue insertion in alpha 2 and only 27% sequence identity between the two homeodomains. Moreover, the orientation of the recognition helix on the DNA is also conserved. This docking arrangement is maintained by side chain contacts with the DNA--primarily the sugar-phosphate backbone--that are identical in alpha 2 and engrailed. Since these residues are conserved among all homeodomains, we propose that the contacts with the DNA are also conserved and suggest a general model for homeodomain-DNA interactions.

Base Sequence↗
PubMed1991

Crystallization and preliminary X-ray diffraction studies of a MAT alpha 2-DNA complex.

Crystals have been obtained of the DNA-binding domain of the yeast MAT alpha 2 repressor bound to a 21 base-pair DNA site. The crystals are grown from polyethylene glycol and CaCl2 and form in space group P2(1) with a = 60.1 A, b = 39.4 A, c = 68.7 A and beta = 98 degrees. They diffract to 2.9 A resolution and contain one protein-DNA complex in the crystallographic asymmetric unit.

Base Sequence↗
PubMed1991

Secondary structure of the homeo domain of yeast alpha 2 repressor determined by NMR spectroscopy.

The yeast alpha 2 protein is a regulator of cell type in Saccharomyces cerevisiae. It represses transcription of a set of target genes by binding to an operator located upstream of each of these genes. The alpha 2 protein shares weak sequence similarity with members of the homeo domain family; the homeo domain is a 60-amino-acid segment found in many eukaryotic transcriptional regulators. In this paper we address the question of whether alpha 2 is structurally related to prototypical members of the homeo domain family. We used solution 1H and 15N nuclear magnetic resonance [NMR] spectroscopy to determine the secondary structure of an 83-amino-acid residue fragment of alpha 2 that contains the homeo domain homology. We have obtained resonance assignments for the backbone protons and nitrogens of the entire 60-residue region of the putative homeo domain and for most of the remainder of the alpha 2 fragment. The secondary structure was determined by using NOE connectivities between backbone protons, 3JHN-H alpha coupling constants, and dynamical information from the hydrogen exchange kinetics of the backbone amides. Three helical segments exist in the alpha 2 fragment consisting of residues 11-23, 32-42, and 46-60 (corresponding to residues 138-150, 159-169, and 173-187 of the intact protein). The positions of these three helices correspond extremely well to those of the Drosophila Antennapedia (Antp) and engrailed (en) homeo domains, whose three-dimensional structures have recently been determined by NMR spectroscopy and X-ray crystallography, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Histochemistry and ultrastructure of the metacercarial cysts of blackspot trematodes Uvulifer ambloplitis and Neascus pyriformis.

Cysts of Uvulifer ambloplitis from green sunfish, Lepomis cyanellus, and Neascus pyriformis from red shiners, Notropis lutrensis, were studied with light-level histochemistry and scanning and transmission electron microscopy. Cysts of both species are bilayered, consisting of an outer host capsule and an inner parasite cyst; the space between these layers is filled with a viscous material. The outer portion of the host capsule of both species is composed of fibrocytes, melanin granules, and collagen fibrils, and the inner portion of layers of flattened fibrocytes. The parasite cyst of U. ambloplitis is formed of 2 layers, an outer dense layer and an inner light layer, whereas the parasite cyst of N. pyriformis is made of 3 layers. A thin outer light-staining layer is present in addition to the 2 layers observed in U. ambloplitis. Results of histochemical staining were the same for both species. The host capsule stained positively for proteins and neutral and acid mucopolysaccharides. The viscous material was positive for neutral and acid mucopolysaccharides but not for proteins. The parasite cyst gave a strong positive reaction for neutral mucopolysaccharides but was negative for acid mucopolysaccharides and proteins.

Animals↗
PubMed1990

Results of treatment of cancer of the cervix in Wellington 1980-4.

A review was undertaken of all 115 women with invasive carcinoma of the cervix referred for treatment to the Wellington regional centre during the period 1980-4. In comparison with the previous five year period, there were 31 (37%) more patients, and a higher proportion of patients had stage I and II disease. The incidence of disease was significantly greater in Maori compared with nonMaori. Treatment was by various combinations of surgery and radiation according to agreed protocols. The actuarial survival at five years was 73% for the whole group, 89% for 64 stage I patients, 65% for 28 stage II patients and 45% for 19 stage III patients. The actuarial risk of a major treatment complication within the first five years was 8.8%. These results confirm that regional centres in New Zealand employing a multidisciplinary approach to patient assessment and treatment can achieve high cure rates with an acceptable incidence of treatment complications. However, prevention of the disease by effective cervical screening programmes should remain an objective of health services in New Zealand.

Adult↗
PubMed1990

Effect of heat shock proteins on survival of isolated aortic cells from normal and atherosclerotic cynomolgus macaques.

Alterations in the level and distribution of arterial heat shock proteins (HSPs) have been demonstrated during atherogenesis. The significance of these changes within the developing and dying plaque, however, is not understood. To examine a potential protective effect of HSPs on arterial cells, enzymatically isolated cells from normal and diet-induced atherosclerotic aortas of cynomolgus macaques were stressed for 20 h with and without added 72/73 kDa heat shock proteins (HSP-72/73), over a range of temperatures and dosages. Cells were analyzed for changes in viability and lysosomal membrane integrity. The responses of cells from normal and diseased aortas for all test regimens were similar. Viability and lysosomal membrane integrity of untreated cells were correlated (r = 0.85), and both factors declined with increasing thermal stress (P less than 0.0005 and P less than 0.004, respectively). Exogenous HSP-72/73 increased cell viability after stress at a minimum concentration of 10 micrograms/ml (P less than 0.05) but was not concentration dependent. Response to different temperatures showed exogenous HSP-72/73 increased cell survival at all temperatures, with the greatest effect at 37 degrees C (P less than 0.01). In contrast, HSP-72/73 did not have an effect on lysosomal membrane integrity for the test period studied. The results demonstrate exogenous HSP-72/73 increases arterial cell survival, suggesting these proteins are associated with a protective mechanism in normal and diseased arteries.

Animals↗
PubMed1990

Accelerated fractionation radiotherapy for advanced head and neck cancer.

Between 1981 and 1986, 89 patients with advanced head and neck squamous cancer were treated with a continuous accelerated fractionation radiotherapy (AFRT) regimen. Three fractions of 1.80 Gy, 4 h apart, were given on three treatment days per week (Monday, Wednesday, Friday), and the tumour dose was taken to 59.40 Gy in 33 fractions in 24-25 days. Acute mucosal reactions were generally quite severe, but a split was avoided by providing the patient with intensive support, often as an in-patient, until the reactions settled. Late radiation effects have been comparable to those obtained with conventional fractionation. The probability of local-regional control was 47% at 3 years for 69 previously untreated patients, whereas it was only 12% at one year for 20 patients treated for recurrence after radical surgery. Fifty-eight previously untreated patients with tumours arising in the upper aero-digestive tract were analysed in greater detail. The probability of local-regional control at 3 years was 78% for 17 Stage III patients and 15% for 31 Stage IV patients. This schedule of continuous AFRT is feasible and merits further investigation.

Carcinoma, Squamous Cell↗
PubMed1990

A statistical approach for the development of an oral controlled-release matrix tablet.

Tablet matrix compositions for optimized prolonged release were selected by surface response methodology. The extreme vertices experimental design was used to develop a surface response model which mathematically defined the release of active component from the tablet matrix as controlled by the percentage of the excipient components. The model, a statistical quadratic equation with a standard error of 3.3, was validated for accurate prediction of drug release profiles and used to identify optimum formulations. This study demonstrated a new application of the extreme vertices experimental design, an efficient method for evaluating a complex mixture system for controlled release, where specific constraints are placed on one or more of the components.

Administration, Oral↗
PubMed1990

H-ras(val12) induces cytoplasmic but not nuclear events of the cell cycle in small Xenopus oocytes.

Microinjection of H-ras(val12) protein into fully grown Xenopus oocytes has been shown to induce meiotic maturation. In the present study, mRNA encoding the mutant ras protein was injected into both fully grown (stage 6) and growing (stage 4) oocytes. The mRNA induced nuclear breakdown in stage 6 oocytes, as expected. However, the mRNA induced neither nuclear breakdown nor maturation promoting factor when injected into stage 4 oocytes. Instead, the response in stage 4 oocytes included an activation pulse of calcium, cortical granule breakdown, elevation of the vitelline envelope, and abortive cleavage furrows, all of which are characteristics of the activation response in mature eggs. In addition, the injected mRNA led to increased rates of endogenous protein synthesis and the migration of subcortical organelles into the oocyte interior. These observations are discussed relative to the suggestion that oncogenic ras protein leads to an increase in both diacylglycerol and inositol trisphosphate, which then regulate the various cytoplasmic events described.

Animals↗
PubMed1990

In vivo regulation of MPF in Xenopus oocytes.

Entry into M phase in the eukaryotic cell cycle is controlled by the oscillating activity of MPF. The active component of MPF is now known to be the p34cdc2 protein kinase originally found in yeast. The p34cdc2 protein kinase displays a characteristic M-phase-specific histone H1 kinase activity when it interacts with cyclins, which are proteins that oscillate through the cell cycle and are thought to regulate p34cdc2 activity. Cyclins can induce M phase when introduced into fully grown Xenopus oocytes and cyclin may play a role in normal oocyte maturation. Small Xenopus oocytes do not mature in response to the hormonal triggers which act on stage 6 oocytes. We introduced cyclin into stage 4 (small) Xenopus oocytes and showed that it activates MPF in these cells, probably by interacting with endogenous p34cdc2 kinase. We made labelled extracts from cyclin-mRNA-injected stage 4 oocytes and used them to show differential stability of clam cyclins A and B at oocyte maturation. The relative stability of the two forms of cyclin related directly to their ability to stabilize crude MPF preparations from injected stage 6 oocytes.

Animals↗
PubMed1990

Subclinical ammonia toxicity in steers: effects on hepatic and portal-drained visceral flux of metabolites and regulatory hormones.

Four calves (avg wt 161 kg) were surgically fitted with indwelling catheters in the femoral artery and femoral, portal, hepatic and mesenteric veins to study the effects of subclinical ammonia toxicity on portal-drained viscera (PDV) and hepatic (HEP) net flux of key metabolites and pancreatic hormones. Hyperammonemia was induced via administration of ammonium chloride (NH4Cl; 12 mumol.kg BW-1.min-1) via the femoral vein catheter for 240 min; infusions were preceded (PRE) and followed (POST) by 60- and 180-min control periods, respectively. Blood samples were obtained from the arterial catheters, and portal and hepatic vein catheters. Net flux rates were calculated by multiplying venoarterial differences by blood flow. Arterial plasma ammonia N peaked (P less than .01) at 327 micrograms/dl; hepatic ammonia extraction increased (P less than .01) from 10 to 23% during NH4Cl infusion. Arterial plasma glucose concentrations increased (P less than .05) during NH4Cl infusion (90.5 vs 82.6 mg/dl) concomitant with trends toward a reduction in net HEP glucose output. Portal-drained visceral release of insulin did not increase (P greater than .10) during NH4Cl infusion despite the steady rise in circulating glucose concentration; however, cessation of NH4Cl infusion resulted in a 109% increase (P less than .05) in PDV insulin release at +60 min POST. Plasma L-lactate, nonesterified fatty acids, urea N and glucagon concentrations and net fluxes were variable throughout the experiment. Results tend to indicate that hyperammonemia reduced hepatic glucose output and glucose-mediated pancreatic insulin release.

Ammonia↗
PubMed1990

The aetiology of bleeding peptic ulcer--as observed in a district general hospital.

A review was made of the hospital case notes of 124 patients admitted with symptoms of upper gastrointestinal haemorrhage due to bleeding peptic ulcer over a five-year period. A high proportion of the patients were found to be elderly and female, as observed by Walt et al 1986. This finding was more pronounced if the source of the bleed was a gastric ulcer. Many of these elderly patients had been on anti-inflammatory drugs at the time of presentation. Concomitant treatment with cimetidine or demulcents did not seem to prevent episodes of bleeding. Many patients on anti-inflammatory drugs bled repeatedly after apparently adequate medical or surgical treatment.

Adult↗
PubMed1989

Yeast repressor alpha 2 binds to its operator cooperatively with yeast protein Mcm1.

To bring about repression of a family fo genes in Saccharomyces cerevisiae called the a-specific genes, two transcriptional regulatory proteins, alpha 2 and GRM (general regulator of matin type), bind cooperatively to an operator found upstream of each a-specific gene. To date, GRM has been defined only biochemically. In this communication we show that the product of a single yeast gene (MCM1) is sufficient to bind cooperatively with alpha 2 to the operator. We also show that antiserum raised against the MCM1 gene product recognizes GRM from yeast cells. These results, in combination with previous observations, provide strong evidence that MCM1 encodes the GRM activity.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

The yeast cell-type-specific repressor alpha 2 acts cooperatively with a non-cell-type-specific protein.

The yeast alpha 2 protein is a cell-type-specific transcriptional repressor. It acts by binding to an operator located upstream of each of its target genes. In this paper, we describe a protein (GRM) that is present in all cell types and binds cooperatively with alpha 2 resulting in an unusual arrangement of the two proteins at the operator. A dimer of alpha 2 occupies the two ends of the operator and straddles the GRM protein, which binds to the center of the operator. Using mutant operators, we show that the recognition sequences for both GRM and alpha 2 are required for repression of a test promoter in vivo. Finally, we deduce that the GRM/alpha 2 cooperativity is mediated through a protein-protein interaction between GRM and the N-terminal domain of alpha 2. This conclusion follows from the observation that the isolated C-terminal domain of alpha 2 can co-occupy the operator with GRM but does not bind cooperatively with GRM.

Base Sequence↗