Biomedical subjects
Publications and source records attributed to R Foster.
An unusual case of Merkel cell carcinoma is presented in which the time course to development of nodal and haematogenous metastases was protracted and the predominant site of metastatic disease was small bowel.
In mitogenically stimulated cells, a specific complex forms between the Ras GTPase-activating protein (RasGAP) and the cellular protein p190. We have previously reported that p190 contains a carboxy-terminal domain that functions as a GAP for the Rho family GTPases. Thus, the RasGAP-p190 complex may serve to couple Ras- and Rho-mediated signalling pathways. In addition to its RhoGAP domain, p190 contains an amino-terminal domain that contains sequence motifs found in all known GTPases. Here, we report that p190 binds GTP and GDP through this conserved domain and that the structural requirements for binding are similar to those seen with other GTPases. While the purified protein is unable to hydrolyze GTP, we detect an activity in cell lysates that can promote GTP hydrolysis by p190. A mutated form of p190 that fails to bind nucleotide retains its RasGAP binding and RhoGAP activities, indicating that GTP binding by p190 is not required for these functions. The sequence of p190 in the GTP-binding domain, which shares structural features with both the Ras-like small GTPases and the larger G proteins, suggests that this protein defines a novel class of guanine nucleotide-binding proteins.
Plant bZIP proteins exhibit a relaxed DNA-binding specificity for DNA sequence motifs containing an ACGT core. Gel mobility shift experiments employing ten different recombinant plant bZIP proteins demonstrated that nucleotides flanking the ACGT core affected binding specificity and identified three different types of ACGT elements: G-box, CACGTG; C-box, GACGTC; and A-box, TACGTA, motifs. These ten different bZIP proteins could be categorized into three groups according to their qualitative and quantitative specificity for G-box and C-box elements. Dissociation constant values (Kd values) of these bZIP proteins for high affinity G-box and C-box elements and reciprocal competition gel mobility shift assays confirmed our classification scheme. Group 1 proteins exhibit a stronger binding affinity for G-box elements, group 2 proteins bind both G-box and C-box motifs with comparable binding affinity, whereas the group 3 proteins display a stronger binding affinity for C-box oligonucleotides. Studies using a panel of G-box and C-box oligonucleotides differing in their flanking sequences identified high affinity binding sites. All ten plant bZIP proteins examined, except TGA1a, exhibited type A G-box binding activity preferring class I G-box elements. In contrast to the situation observed for G-box elements, C-box motifs displayed a very much more stringent flanking nucleotide requirement for binding activity. Protein/DNA binding experiments using scanning mutants of a high affinity G-box element and G-box/C-box hybrid elements demonstrated that bZIP protein binding activity depends upon the affinity of protein dimer subunits for ACGT half-sites. Information provided by our systematic analysis of plant bZIP DNA binding specificity can be used to identify high affinity binding sites for the plant bZIP proteins studied here. Assuming that only high affinity bZIP binding sites are likely to function in vivo, identification of these sites will allow us to predict which genes are activated by a particular bZIP protein.
A thin-layer twin-electrode electrochemical cell in which the working and auxiliary electrodes are facing each other and which contains the appropriate enzyme and a mediator was used for the determination of cholesterol or glucose. A steady-state diffusion-controlled response was obtained for cholesterol whereas for glucose the response was limited by diffusion and kinetics. Simulation of the system showed that a steady-state response is obtained, in the absence of kinetic complications, after a time of 0.01 d2/Ds, where d is the distance between the two electrodes and D is the diffusion coefficient of the mediator. Linear plots of steady-state current versus concentration were obtained for cholesterol and glucose. The results were compared with those expected theoretically and a thin-layer capillary fill device is proposed.
The West Virginia School of Osteopathic Medicine (WVSOM) educated and retained more primary care physicians for practice in rural Appalachia than did any other U.S. medical school from 1978 through 1990. This article describes the most important methods used at WVSOM to place physicians in rural areas: (1) The school has a focused, achievable mission (to provide primary care physicians who are trained to meet the medical needs of rural Appalachia and to improve the health care of the rural Appalachian population) that is agreed upon by the administration, faculty, and students; (2) it participates in a multistate educational exchange program with a similar mission; (3) it emphasizes personalized and interactive recruiting, admission, and placement processes aimed to attract nontraditional, rural students; (4) it provides early and long-term clinical training in rural sites (both hospitals and physicians' offices); (5) it is dedicated primarily to the education of medical students rather than to research or other goals; and (6) it is a freestanding school in a rural environment. The authors state that although WVSOM is unusual in some respects, at least some of its methods may be useful to other medical schools as they seek to produce more primary care physicians for rural and other underserved areas.
The Saccharomyces cerevisiae SWI4 gene encodes an essential transcription factor which controls gene expression at the G1/S transition of the cell cycle. SWI4 transcription itself is cell cycle regulated, and this periodicity is crucial for the normal cell cycle regulation of HO and at least two of the G1 cyclins. Since the regulation of SWI4 is required for normal cell cycle progression, we have characterized cis- and trans-acting regulators of SWI4 transcription. Deletion analysis of the SWI4 promoter has defined a 140-bp region which is absolutely required for transcription and can function as a cell cycle-regulated upstream activating sequence (UAS). The SWI4 UAS contains three potential MluI cell cycle boxes (MCBs), which are known cell cycle-regulated promoter elements. Deletion of all three MCBs in the SWI4 UAS decreases the level of SWI4 mRNA 10-fold in asynchronous cultures but does not abolish periodicity. These data suggest that MCBs are involved in SWI4 UAS activity, but at least one other periodically regulated element must be present. Since SWI6 is known to bind to MCBs and regulate their activity, the role of SWI6 in SWI4 expression was analyzed. Although the MCBs cannot account for the full cell cycle regulation of SWI4, mutations in SWI6 eliminate the normal periodicity of SWI4 transcription. This suggests that the novel cell cycle-regulated element within the SWI4 promoter is also SWI6 dependent. The constitutive transcription of SWI4 in SWI6 mutant cells occurs at an intermediate level, which indicates that SWI6 is required for the full activation and repression of SWI4 transcription through the cell cycle. It also suggests that there is another pathway which can activate SWI4 transcription in the absence of SWI6. The second activator may also target MCB elements, since SWI4 transcription drops dramatically when they are deleted.
One goal of supportive housing is to enlarge and improve the functioning of the social support networks of residents. The networks of a convenience sample of 42 residents were assessed using scales developed by Barrera. The size of the networks (11.5) was no larger than that reported for similar clients living in other types of community settings but the composition differed. Staff and co-residents appear to partially replace rather than add to family and friends. This is of concern because friends are uniquely important determinants of satisfaction. An association between perceived need for support and network size was also found. These findings suggest some positive influences but also raise questions about unintended negative consequences of living in artificially constructed social environments.
We have analyzed a host- and temperature-dependent mutant of Rous sarcoma virus in order to learn more about the nature of mutations which lead to a host range phenotype. We have cloned and sequenced the v-src genes from this mutant, tsLA33-1, and from its presumed parent, tsLA33. Both the tsLA33 and the tsLA33-1 pp60v-src proteins contain multiple mutations. The tsLA33 v-src gene product has amino acid alterations at four positions. In the tsLA33-1 v-src gene product, two of these four mutations have reverted to wild type. We have constructed chimeras between the two mutant v-src gene products and between each mutant and the Prague A v-src gene product. To assess the contribution of each amino acid change to the transformation phenotypes of tsLA33 and tsLA33-1, we expressed the hybrid proteins in both chicken embryo fibroblasts and Rat-3 fibroblasts. Additionally, we have measured the protein tyrosine kinase activity of chimeras constructed between the tsLA33 and tsLA33-1 pp60v-src proteins. Our results indicate that mutations in the catalytic domain of each protein are the principal determinants of the transforming ability and protein tyrosine kinase activity of the tsLA33 and tsLA33-1 pp60v-src proteins.
The CACGTG G-box motif is a highly conserved DNA sequence that has been identified in the 5' upstream region of plant genes exhibiting regulation by a variety of environmental signals and physiological cues. Gel mobility shift assays using a panel of G-box oligonucleotides differing in their flanking sequences identified two types of binding activity (A and B) in a cauliflower nuclear extract. Competition gel retardation assays demonstrated that the two types of binding activity were distinct. Type A binding activity interacted with oligonucleotides designated as class I elements, whereas type B binding activity interacted strongly with class II elements and weakly with class I elements. A third class of elements, null elements, did not exhibit any detectable binding under our assay conditions. Gel retardation analysis of nonpalindromic hybrid G-box oligonucleotides indicated that hybrid elements of the same class exhibited binding affinity commensurate with the affinity of the weaker element, hybrid class I/II elements exhibited only type B binding, and hybrid class I/null and class II/null elements did not show any detectable binding activity. These binding activities can be explained by the affinity of bZip G-box binding homo- or heterodimer subunits for G-box half sites. These experiments led to a set of classification rules that can predict the binding activity of all reported plant G-box motifs containing the consensus hexameric core. Tissue- and/or development-specific expression of genes containing G-box motifs may be regulated by the affinity of G-box proteins for the different classes of G-box elements.
The current debate about how to provide housing for persons with persistent psychiatric disabilities should be informed by empirical information from a variety of sources. Data from a study of 17 residents discharged from Regeneration House Inc., illustrates the value of consumer perceptions in the evaluation of supportive housing. Extensive exploration of the positive and negative aspects of living in a group home from the perspective of experienced residents provides useful input for programme planning. The implications of the findings for the individual programme and the wider field are discussed.
STUDY OBJECTIVE: To assess the accuracy of the respiratory inductive plethysmography in the measurement of PEEP-induced changes in end-expiratory lung volume during mechanical ventilation and its accuracy and stability in the measurement of ventilation during controlled mechanical ventilation and spontaneous breathing. DESIGN: An open comparison between two methods using a criterion standard. Either a pneumotachometer (mechanically ventilated patients) or a spirometer (spontaneously breathing subjects) was used as the reference method. SETTING: Tertiary care center; a multidisciplinary intensive care unit and a metabolic research unit. PATIENTS: Six mechanically ventilated, paralyzed postoperative open heart surgery patients, six spontaneously breathing COPD patients, and eight healthy volunteers. INTERVENTIONS: Stepwise increases and reductions of PEEP from zero to 12 cm H2O during controlled mechanical ventilation; repeated validation of the calibration of the respiratory inductive plethysmography (RIP) in both mechanically ventilated and spontaneously breathing subjects. MEASUREMENTS AND RESULTS: The baseline drift of the RIP in vitro was 10 ml/150 min and in a ventilated model it was 20 ml/150 min. In mechanically ventilated patients, the mean error of the calibration after 150 min was within +/- 5 percent. Change in end-expiratory lung volume (EELV) during the stepwise increase of PEEP up to 12 cm H2O was 849 +/- 136 ml with the RIP and 809 +/- 125 ml with the pneumotachometer (PT), and during the stepwise reduction of PEEP it was 845 +/- 124 ml and 922 +/- 122, respectively (not significant [NS]. The mean difference between methods in the measurement of change in EELV was -6.6 +/- 3.5 percent during increasing and 6.6 +/- 6.7 percent during decreasing PEEP (NS). Both in mechanically ventilated and spontaneously breathing subjects, the difference between methods was significant for VT and VT/TI. The difference in VT was -2.2 +/- 0.2 percent during mechanical ventilation, -1.1 +/- 0.5 percent in spontaneously breathing COPD patients, and 2.9 +/- 0.4 percent in healthy volunteers (NS between groups). CONCLUSIONS: The RIP is sufficiently accurate for the measurement of PEEP-induced changes in EELV during controlled mechanical ventilation. The accuracy of tidal volume measurement is similar during mechanical ventilation and spontaneous breathing. The calibration of the RIP is stable enough for bedside monitoring of changes in lung volumes.
The sequences responsible for heavy metal-inducible expression are situated within the proximal 437 and 160 base pairs (bp) of MT-IF and MT-IG 5'-flanking sequence, respectively. Only 105 bp of proximal MT-IG 5'-flanking sequence containing a TATA box, two metal responsive elements (MREs), and three GC motifs and 147 bp of proximal MT-IF 5'-flanking sequence containing a TATCA box, four MREs, and two GC motifs were required for heavy metal-inducible expression. However, the proximal 111 bp of MT-IF 5'-flanking sequences (a TATCA box, two MREs, and two GC motifs) was not responsive to heavy metals and competes less efficiently than the 105-bp MT-IG fragment in a competition transfection analysis. The MT-IF promoter fragment containing MREc and MREd is substantially stronger and a more efficient competitor than the MT-IG promoter fragment containing MREc and MREd. Furthermore, the proximal 160 bp of MT-IG 5'-flanking sequence functions as a strong metal-inducible promoter but not as a metal-inducible enhancer. Mobility shift analysis of MT-IF and MT-IG promoter subregions suggests a correlation between protein binding to MRE sequences and MT gene expression. These data illustrate that the overall structural and functional organization of the MT-IF and MT-IG promoters are very different and that the molecular mechanisms governing differential expression levels of human MT genes are quite complex.
In this study we have demonstrated the ability of 5-azacytidine to elevate the basal level expression of the metallothionein (MT)-IF and MT-IG genes and increase the basal level expression of the MT-IIA gene in Hep G2 cells, a cell line which exhibits heavy metal inducible MT gene expression. Atomic absorption analysis of 5-azacytidine treated Hep G2 cells detected a 2-fold increase in the total cellular copper content. Pretreatment of 5-azacytidine exposed cells with hydroxyurea and cycloheximide indicated that the increase in total cellular copper content was a direct response to 5-azacytidine treatment. S1 nuclease analysis illustrated that pretreatment of Hep G2 cells with KCN, a copper specific chelator and uptake inhibitor, suppressed 5-azacytidine- and copper-inducible MT-IG gene expression. Thus, the increase in MT gene expression in response to 5-azacytidine treatment can be correlated to an increase in the total cellular copper content. Possible mechanisms on how 5-azacytidine could alter the influx/efflux of copper in Hep G2 cells are discussed.
A method of centralizing management of antegrade and retrograde blood cardioplegia delivery at the heart-lung machine is described. Operation of a manually flow-directed pump switch, controlled by the perfusionist, improves safety and efficiency.
Five fresh cadaver upper extremities were studied with use of a static positioning frame, pressure-sensitive film, a microcomputer-based videodigitizing system, and a Sun station image analysis system to assess the load bearing characteristics of the scaphoid in the proximal carpal joint. Specimens were studied in their normal condition, after a proximal pole osteotomy of the scaphoid, and after resection of the proximal pole of the scaphoid. The amount of contact area born through the scaphoid fossa was essentially the same whether the scaphoid was intact, or after a simulated scaphoid fracture of its proximal pole, or after resection of the proximal pole. The scaphoid contact area and pressure, although overall relatively constant, was redistributed after osteotomy, resulting in increased contact area under the distal fragment and no change or a slight decrease in the contact area under the proximal fragment of the scaphoid. After resection of the proximal fragment, all scaphoid contact area and pressure was born by the distal scaphoid fragment. The contact area and pressure characteristics of the lunate remained unchanged in all conditions compared with the normal condition. There were no significant changes in the locations of the centroids of the scaphoid segments and the lunate in any of the conditions tested.
Five fresh cadaver upper extremities were studied by use of a static positioning frame, pressure-sensitive film, and a microcomputer-based videodigitizing system, to assess the load-bearing characteristics of a scaphoid silicone implant within the radioulnarcarpal joint. Specimens were studied in their "normal" condition, after resection of the scaphoid, after placement of a scaphoid implant, and with a scaphoid implant and a simulated capitate-lunate-triquetrum-hamate fusion. The scaphoid silicone implant bore significant, although less, load than the normal scaphoid. Decreasing the size of the scaphoid implant decreased the load born by the implant. Decreased load through the scaphoid implant was compensated by the lunate. The addition of a limited carpal fusion did not significantly decrease the load born by a scaphoid implant. Therefore, the silicone scaphoid implant is a load-bearing implant even when undersized or placed in association with a limited carpal fusion.
Cells persistently spread on OPN or collagen survive heat shock better than cells transiently spread on fibronectin or tissue culture plates. Thus, a central question is whether constitutively or inducible stress proteins are enhanced in cells grown on adhesive proteins that maintain a persistent spread cell shape. Levels of Hsp 72,73, and colligin/Hsp47 were determined by Western blot analyses. The inducible Hsp 72 was prominently expressed following heat shock in cells grown on OPN or collagen, but not in cells plated on fibronectin coated substratum or on tissue culture plates. Colligin/Hsp 47 and Hsp 73 manifested a similar pattern of expression indicating that these adhesive attachment proteins accommodate cell function through organization of cell architecture.
The metallothionein (MT) gene expression profile was followed in rainbow trout during early embryo development and in liver and gonads during the period of sexual maturation. The hepatic MT mRNA levels increase at the end of sexual maturation in both male and female rainbow trout. Although both isoforms of MT mRNA accumulate in the liver, there is a preferential increase in MT-A in the female liver. Concomitantly with this increase in MT there is a redistribution of zinc and copper to MT. In the juvenile female there is an abundance of MT mRNA in the ovaries. This is correlated to high levels of zinc in the MT fraction upon Sephadex G-75 chromatography. During ovary development the MT mRNA levels and the MT-bound zinc levels drop, with an increase in zinc being bound to high-molecular-mass proteins. At ovulation most of the zinc is found in the membrane portion upon centrifugation. In contrast to the ovaries, there are no apparent changes in either trace metal distribution or MT mRNA levels during testis development. In the developing embryo there is an increase in MT-bound copper at gastrulation. This is accompanied by an increase in both isoforms of MT mRNA. At hatch both the copper and zinc levels increase in the MT fraction, with a concomitant increase in mainly MT-A mRNA. These findings indicate that the variations in MT mRNA levels during development are closely associated with metal regulation.