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Biomedical subjects

S L Walker

Publications and source records attributed to S L Walker.

At least 55 records · Page 3Linked to original sources
PubMed1998

A single exposure of solar simulated radiation suppresses contact hypersensitivity responses both locally and systemically in humans: quantitative studies with high-frequency ultrasound.

Ultraviolet radiation (UVR)-induced suppression of cutaneous cell-mediated immunity plays an important role in the development of photocarcinogenesis in the mouse and a similar role is suspected in humans. Cell-mediated immunity is readily tested in vivo by measuring the contact hypersensitivity (CHS) response to topically applied haptens. CHS in humans is usually determine clinically, with a subjective scoring system. However, these subjective scores cannot be statistically analysed. This paper compares four methods currently used to quantify CHS elicitation responses in humans. The data show that ultrasound images provide the most accurate and reproducible measurements of the clinically observed CHS response. We also demonstrate that assessment of the primary allergic response is a useful indicator of the magnitude of the elicitation response and can be used to avoid severe CHS reactions in volunteers. There are few human studies investigating the effects of solar simulated radiation (SSR) exposure on immunosuppression. In this study we demonstrate SSR is highly immunosuppressive in all subjects tested. Irradiating a small area of skin with a single exposure to 3MEDs of SSR completely suppressed CHS both locally (12/12 volunteers) and systemically (10/12 volunteers). Our data do not support a role for a genetic susceptibility to UVR-induced immunosuppression in humans.

Adolescent↗
PubMed1997

The Rep68 protein of adeno-associated virus type 2 increases RNA levels from the human cytomegalovirus major immediate early promoter.

The Rep68 and Rep78 proteins of adeno-associated virus type-2 (AAV) are multifunctional DNA binding proteins which are involved in the positive and negative regulation of AAV genes, as well as various cellular and heterologous viral genes. In this study we report that Rep68 enhances expression from the major immediate early promoter (MIEP) of human cytomegalovirus (HCMV). This Rep-mediated enhancement of RNA levels is abrogated by the introduction of a Rep recognition sequence (RRS) at either position -18 or -244 in the HCMV-MIEP. However, a mutant RRS (mRRS), which is not bound by Rep68 is unable to negate the effect of Rep68. Sequence analysis and electrophoretic mobility shift assays showed no Rep68 binding sites within the wild-type HCMV-MIEP. Rep68 may therefore be enhancing expression from the HCMV-MIEP by interacting with other regulatory proteins that have an effect on the expression from this promoter or by altering the expression of a cellular gene whose product influences the HCMV-MIEP. Our results may also help to explain the previous observation that coinfection with AAV enhances the cytopathic effect of HCMV.

Base Sequence↗
PubMed1997

Sunscreens offer the same UVB protection factors for inflammation and immunosuppression in the mouse.

Many studies report that sunscreens effective against UVR-induced inflammation afford poor protection against immunosuppression. We have studied the relationship between photoprotection of inflammation and immunosuppression with monochromatic UVB (Philips TL01 tubes, lambda max = 311 nm) to remove possible confounding effects of differences in end point action spectra. Dose-response curves for edema and systemic suppression of contact hypersensitivity (CHS) in HRA.HRII-c/+/Skh mice showed that suppression of CHS was more sensitive to UVB irradiation by a factor of 2. The UVB dose-response curve for murine edema was similar to that for human erythema, with threshold doses of 773 mJ x cm(-2) and 632 mJ x cm(-2), respectively. The protection afforded by two UVB filters, octyl dimethyl para-aminobenzoic acid and 2-ethylhexyl-4'-methoxycinnamate, prepared in an identical vehicle, each with the same optical density at 311 nm, was tested in mice. We applied sunscreen to all exposed skin or to transpore tape above the irradiation cages, prior to exposure with 2.8 minimal edema doses. Topical or tape application of both sunscreens protected totally against edema but only partially against immunosuppression, with no significant difference in protection between the two application techniques (p > 0.4). A sunscreen protection factor of 4 in vivo was determined for 2-ethylhexyl-4'-methoxycinnamate for both edema and immunosuppression. Failure of the sunscreens to protect completely against immunosuppression was due to the ability of subedemal doses of UVB to induce substantial immunosuppression and not, as previously suggested, to any skin interaction.

Animals↗
PubMed1997

Mutational analysis of the adeno-associated virus Rep68 protein: identification of critical residues necessary for site-specific endonuclease activity.

The Rep68 and Rep78 proteins of adeno-associated virus type 2 (AAV) are multifunctional proteins which contain overlapping amino acid sequences. They are required for viral replication and preferential integration of the AAV genome into a region of human chromosome 19. During the terminal resolution process of AAV DNA replication, these proteins make a site-specific and strand-specific endonuclease cut within the AAV inverted terminal repeat DNA. The Rep68 and Rep78 proteins also have helicase and DNA-binding activities. The endonuclease activity is believed to involve the covalent attachment of Rep68 or Rep78 at the cut site via a phosphotyrosine linkage. In an attempt to identify the active-site tyrosine residue of Rep78 and Rep68, tyrosine residues were site specifically mutated to phenylalanines by overlap extension PCR, and the resulting PCR fragments were cloned into a maltose binding protein-Rep68 fusion (MBP-Rep68delta) expression vector. The mutant MBP-Rep68delta proteins were expressed in Escherichia coli cells, purified with amylose resin, and assayed in vitro for Rep68-specific activities. Although several of the mutations disrupted the endonuclease activity, only the mutation of tyrosine 152 abrogated the endonuclease activity with no discernible effect on the helicase or DNA-binding activities. Our data therefore suggest that there are distinct active sites for the helicase and endonuclease activities.

ATP-Binding Cassette Transporters↗
PubMed1997

Mutational analysis of the adeno-associated virus type 2 Rep68 protein helicase motifs.

The adeno-associated virus type 2 (AAV) Rep78 and Rep68 proteins are required for viral replication. These proteins are encoded by unspliced and spliced transcripts, respectively, from the p5 promoter of AAV and therefore have overlapping amino acid sequences. The Rep78 and Rep68 proteins share a variety of activities including endonuclease, helicase, and ATPase activities and the ability to bind AAV hairpin DNA. The part of the amino acid sequence which is identical in Rep78 and Rep68 contains consensus helicase motifs that are conserved among the parvovirus replication proteins. In the present study, we mutated highly conserved amino acids within these helicase motifs. The mutant proteins were synthesized as maltose binding protein-Rep68 fusions in Escherichia coli cells and affinity purified on amylose resin. The fusion proteins were assayed in vitro, and their activities were directly compared to those of the fusion protein MBP-Rep68 delta, which contains most of the amino acid sequences common to Rep78 and Rep68 and was demonstrated previously to have all of the in vitro activities of wild-type Rep78 and Rep68. Our analysis showed that almost all mutations in the putative helicase motifs severely reduced or abolished helicase activity in vitro. Most mutants also had ATPase activity less than one-eighth of the wild-type levels and lacked endonuclease activity.

ATP-Binding Cassette Transporters↗
PubMed1997

The effects of growth dynamics upon pH gradient formation within and around subsurface colonies of Salmonella typhimurium.

pH measurements made in and around submerged colonies of Salmonella typhimurium grown within a model gelatin gel system using pH-sensitive micro- and macroelectrodes indicated some pH heterogeneity occurring in and around the bacterial colony. Inoculation density, initial pH and glucose concentration were all found to influence colony diameter and metabolism of Salmonella colonies. Colony growth in the presence of glucose, at pH 7.0 with an inoculation density of 1 cell ml-1 led to a pH fall of 1-2 pH units after 2 d. At pH 5.0, with glucose, colony growth rates were much slower than at pH 7.0, and the pH change varied by less than one pH unit often becoming alkaline. In the absence of glucose, only small pH changes were observed within the medium, although growth rates were similar to those in glucose-containing media. At the higher inoculation density (ca 1000 cells ml-1), isolated pH changes were not observed. Morphological changes, such as the production of annular rings, were noted in stationary phase colonies as was alkali production in colonies. These results are discussed in relation to observations with surface colonies.

Culture Media↗
PubMed1996

L-DOPA/carbidopa for nocturnal movement disorders in uremia.

Restless legs syndrome (RLS) and periodic limb movements during sleep (PLMS) are sleep disorders that are common and distressing to uremic patients. There are few data regarding effective treatment in this population. Five chronic hemodialysis patients completed a double-blind, placebo-controlled, crossover study using a single bedtime dose of controlled release L-DOPA/carbidopa (100/25 mg) for treatment of RLS and sleep disruption. Leg movements per hour of sleep and percentage of sleep time accompanied by leg movements were decreased with treatment (101.0 +/- 29.1 events/hour on placebo vs. 61.0 +/- 28.3 events per hour on drug, p = 0.006; and 15.1 +/- 4.9% of sleep time with leg movements on placebo vs. 8.6 +/- 4.0% on drug, p = 0.014). In addition, arousals associated with leg movements (mean 209 +/- 49 events on placebo, mean 108 +/- 46 events on drug) and the leg movement arousal index (mean 59 +/- 23 events/hour on placebo, mean 23 +/- 9 events/hour on drug) were decreased by active medication (p = 0.03 and 0.04, respectively). Patients, however, continued to have very disrupted sleep and we could not document consistent subjective or objective improvement in overall sleep except for an increase in slow-wave sleep (SWS) from 9.0% to 22.8% (p = 0.01). The patterns of movements during sleep were not uniform in different patients, and the movements, although often periodic, were much longer than defined for PLMS. Because of this, finding suitable objective parameters to analyze was problematic. Measuring the percentage of sleep time during which there were leg movements was probably the most efficient and reproducible means of quantitating this disorder. Thus, although controlled-release L-DOPA/carbidopa at a dose of 100/25 mg given once nightly reduced leg movements and increased SWS, sleep continued to be disrupted. Whether higher doses or more frequent dosing is effective requires further investigation.

Aged↗
PubMed1995

ToxR (RegA) activates Escherichia coli RNA polymerase to initiate transcription of Pseudomonas aeruginosa toxA.

The Pseudomonas aeruginosa (Pa) structural gene (toxA), which encodes the exotoxin A protein has been shown to be regulated at the transcriptional level by a protein designated ToxR (also known as RegA). We have previously reported that ToxR directly enhances toxA transcription in vitro; however, in the absence of ToxR, Pa RNA polymerase (RNAP) transcribes toxA with low efficiency. In the present study, we have examined the ability of ToxR to initiate toxA transcription using the heterologous Escherichia coli (Ec) RNAP and found that ToxR can function with Ec RNAP to efficiently transcribe toxA both in vitro and in vivo. Antibodies produced against the sigma 70 subunit of Ec RNAP inhibit ToxR-mediated enhancement of toxA transcription, suggesting that the RNAP holoenzyme (E sigma 70) is required for transcriptional activation of toxA. We further demonstrate that ToxR is required for open-complex formation at the toxA promoter. By selectively deleting toxA upstream sequences, we have localized at 214-bp region containing both the toxA promoter and a putative ToxR-binding site sufficient for ToxR-mediated transcription of toxA.

ADP Ribose Transferases↗
PubMed1995

Effects of stromelysin activity on proteoglycan degradation of canine articular cartilage explants.

We investigated whether stromelysin activity in the medium of canine articular cartilage explants is associated with proteoglycan degradation in these explants. Cartilage explants were treated with recombinant human interleukin 1 alpha (rh-IL-1 alpha), lipopolysaccharide, or canine monocyte-conditioned medium. Proteoglycan synthesis and degradation were measured. Metalloproteinase activity (inhibitable by tissue inhibitor of metalloproteinase 2) in the culture medium was measured by use of fluorimetry with a quenched fluorescent substrate. Western blots of the medium were probed with polyclonal antibodies to human stromelysin, collagenase, and gelatinase. Neither metalloproteinase activity nor proteoglycan degradation were inducible in canine cartilage explants treated with rh-IL-1 alpha. However, proteoglycan synthesis was significantly (P < 0.05) decreased by concentrations of 10 and 100 ng of rh-IL-1 alpha/ml. Metalloproteinase activity in the medium accompanied proteoglycan degradation of cartilage treated with lipopolysaccharide and monocyte-conditioned medium. The metalloproteinase released into the medium was identified as prostromelysin by results of western blotting.

Animals↗
PubMed1994

ToxR (RegA)-mediated in vitro transcription of Pseudomonas aeruginosa toxA.

Exotoxin A (ETA) has been described as a major virulence factor produced by the opportunistic pathogen Pseudomonas aeruginosa. The transcription of the ETA structural gene (toxA) has been shown to be positively regulated by the product of the toxR gene (also called regA). However, the mechanism by which ToxR regulates toxA transcription is still under investigation. We have expressed toxR in Escherichia coli under the control of the T7 promoter and purified the wild-type ToxR protein. We have also produced ToxR as a fusion protein consisting of the first 12 amino acids of the T7 capsid protein attached to the N terminus of the intact ToxR protein. In the present study we have developed and used an in vitro transcription assay in order to investigate the mechanism of ToxR-mediated transcriptional regulation of toxA. Under the conditions of this in vitro assay toxA transcription requires the toxR product in addition to P. aeruginosa RNA polymerase (RNAP). Both the native and the T7::ToxR fusion proteins facilitate initiation of toxA transcription in vitro in the presence of Pseudomonas RNAP. Additional studies using (i) specific enzyme-linked immunosorbent assay; (ii) indirect immunoprecipitation; and (iii) gel-filtration chromatography, indicate that ToxR binds to the purified Pseudomonas RNAP and strengthens the possibility that ToxR may be an alternative sigma factor. Furthermore, the ToxR-mediated transcription of toxA is increased approx. threefold in the presence of crude cytoplasmic extracts from P. aeruginosa ToxR+ or ToxR-RegB- strains, indicating that additional factors play a role in the efficient and optimal transcription of toxA.

ADP Ribose Transferases↗
PubMed1994

Relationship between the ability of sunscreens containing 2-ethylhexyl-4'-methoxycinnamate to protect against UVR-induced inflammation, depletion of epidermal Langerhans (Ia+) cells and suppression of alloactivating capacity of murine skin in vivo.

The UVB sunscreen 2-ethylhexyl-4'-methoxycinnamate was evaluated in hairless albino mouse skin for its ability to inhibit UVR-induced (i) oedema, (ii) epidermal Langerhans cell (Ia+) depletion and (iii) suppression of the alloactivating capacity of epidermal cells (mixed epidermal cell-lymphocyte reaction, MECLR). The sunscreen, prepared at 9% in ethanol or a cosmetic lotion, was applied prior to UVB/UVA irradiation. In some experiments there was a second application halfway through the irradiation. Single applications in both vehicles gave varying degrees of protection from oedema and Langerhans cell depletion but afforded no protection from suppression of MECLR. When the sunscreens were applied twice there was improved protection from oedema and Langerhans cell depletion and complete protection was afforded from suppression of MECLR. There was a clear linear relationship between Langerhans cell numbers and oedema with and without sunscreen application. The relationship between Langerhans cell numbers and MECLR was more complex. These data confirm published discrepancies between protection from oedema (a model for human erythema) and endpoints with immunological significance, but show that 2-ethylhexyl-4'-methoxycinnamate can afford complete immunoprotection, although protection is dependent on the application rate and vehicle.

Administration, Topical↗
PubMed1993

Comparison of efficacy of salbutamol and sodium cromoglycate in the prevention of ticarcillin-induced bronchoconstriction.

Nebulized ticarcillin can cause bronchoconstriction in children with cystic fibrosis (CF). We assessed whether pretreatment with salbutamol or sodium cromoglycate (SCG) would prevent this side-effect using a randomized, double-blind, placebo-controlled design. Fifteen children with CF received pretreatments of saline, SCG, or salbutamol, in random order, one on each day. Baseline lung function was measured before and after pretreatment, and after ticarcillin nebulization. On the control day (saline pretreatment), ticarcillin caused a reduction in forced expiratory volume in one second (FEV1), which was maximal 10 minutes after receiving the aerosol and persisted for 120 minutes. The mean maximal fall in FEV1 was 9%. Pretreatment with salbutamol abolished the fall in FEV1 seen with ticarcillin at all time points. Pretreatment with SCG diminished the maximal fall in FEV1 at 10 minutes (mean, 4%) and resulted in the FEV1 returning to baseline within 120 minutes. These data suggest that pretreatment with salbutamol is more effective in preventing ticarcillin-induced bronchoconstriction in the doses used in routine clinical practice, than it is with SCG.

Adolescent↗
PubMed1991

Sunscreens with low sun protection factor inhibit ultraviolet B and A photoaging in the skin of the hairless albino mouse.

We examined the chronic effect of long daily suberythemal, fluorescent solar-stimulated radiation (FSSR; ultraviolet B (UVB)+A(UVA)) and UVA alone on female Skh-1 hairless albino mouse skin. Mice were dorsally irradiated 8 h every weekday for 16 weeks with FSSR or UVA, or 32 weeks with UVA alone. Various topical, low concentration, UVB and/or UVA sunscreens were applied before irradiation. Damage was assessed by skin-fold thickness, histology and biochemically by changes in the proportion of type III collagen. All FSSR-exposed mice showed increased skin thickening, elastic fibre hyperplasia, collagen damage and an increased proportion of type III collagen. Application of the UVB sunscreen (2.00%) resulted in marked protection for all nonbiochemical endpoints. There was no obvious advantage of adding 0.75% UVA sunscreen to the UVB sunscreen, but adding 2.00% UVA sunscreen reduced biochemical changes and connective tissue damage. Sixteen weeks of UVA irradiation caused skin thickening and laxity but the histology and biochemistry were indistinguishable from the controls. The mice irradiated with UVA for 32 weeks showed slight elastic fibre hyperplasia and collagen damage histologically, and increased skin thickening and laxity; these changes were unmodified by the 0.75% UVA sunscreen. These mice showed a significant increase in the proportion of type III collagen against which the UVA sunscreen offered protection. Our data suggest that UVA may be important in photoaging and that the use of low sun protection factor UVB+ UVA sunscreens on a day-to-day basis may offer some protection from solar photoaging.

Administration, Cutaneous↗
PubMed1990

Phototumorigenesis studies of 5-methoxypsoralen in bergamot oil: evaluation and modification of risk of human use in an albino mouse skin model.

The skin of the female hairless albino mouse (Skh 1) was used to study the enhancement of solar simulated radiation (SSR) tumorigenesis by 5-methoxypsoralen (5-MOP) in model perfumes that contain bergamot oil. This work was done in association with yeast mutagenicity studies and human skin phototoxicity studies. Analyses of time-to-onset of tumour observation with 5-MOP at 0, 5, 15 and 50 ppm show a highly significant 5-MOP dose effect and the data indicate that 5-MOP has phototumorigenic potential even at 5 ppm. The addition of 0.5% UVB and 0.5% UVA sunscreens significantly reduces the tumorigenicity associated with the vehicle (i.e. 5-MOP at 0 ppm) and 5-MOP at all concentrations. Pairwise comparisons of 5-MOP (at 5 or 15 ppm) plus sunscreens with vehicle plus sunscreens show that the sunscreens afford total protection at the lower 5-MOP concentrations. Additional studies show that a 5-6 h delay between 5-MOP application and SSR exposure defers the time-to-onset of tumours as does intermittent 5-MOP and SSR treatment. A comparison of 5-MOP at 50 ppm in bergamot oil with 5-MOP at 50 ppm prepared from pure 5-MOP crystals shows identical results, indicating that the active phototumorigenic agent in bergamot oil is 5-MOP and not other related compounds, which may be present at greater concentrations. Analyses of tumour histology at death show, in general, similar patterns of malignancy for all groups. Thus although it is possible to delay tumorigenesis by various strategies, the tumours that eventually develop are just as likely to be malignant, if not more so, when compared with non-delayed groups.

5-Methoxypsoralen↗
PubMed1989

Lumbar zygapophyseal joint meniscoids: evidence of their role in chronic intersegmental hypomobility.

Meniscoid bodies of the zygapophyseal joints are considered by some to be a cause of the Acute-Locked Back Syndrome. These meniscoid bodies have been identified anatomically and histologically, but not yet in a formal clinical investigation. The authors provide a brief review of the zygapophyseal meniscoid-related literature, present new documentation of histological variations and offer some possible consequences of the presence of mechanically pathological meniscoids. It is herein proposed that an important sequela to the pathophysiology of these structures is that of chronic intersegmental hypomobility. We invite the scientific community to pursue further investigations of these meniscoids. Magnetic resonance imaging is one method suggested to investigate the meniscoid in vivo.

Aged↗
PubMed1988

A first approach to an action spectrum for 8-MOP phototumorigenesis in mice.

Groups of hairless albino mice were treated with 8-MOP and exposed to a fixed daily dose of UVR obtained from one of four different emission spectra. Each spectrum was obtained by filtering a xenon arc source with a different WG filter. The resultant spectra showed differences in their proportions of shorter UVA wavelengths. A decreased proportion of the shorter wave bands systematically increased the time to first tumor. These data suggest that the action spectrum for 8-MOP sensitized phototumorigenesis is similar to that of acute 8-MOP action spectra in mammalian skin, which usually show maximal activity in the 320-335 nm region.

Animals↗
PubMed1976

A comparison of two impact noise measurement techniques.

An impact noise analyzer has been tested to determine its accuracy and precision. Results showed that peak estimation when corrected by a suitable correction factor were good and the confidence limits on the corrected peak determination were less than +/- 1.0 dB. Determination of pulse duration with the meter proved to be poor. At present it is useful for making only general estimations of duration. For exact pulse duration measurements, it appears that development of a small, portable oscilloscope holds the most promise for a field instrument with the necessary accuracy and precision.

Environmental Pollution↗