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Biomedical subjects

M A Hughes

Publications and source records attributed to M A Hughes.

At least 19 recordsLinked to original sources
PubMed2006

Evaluation of hypochlorous acid washes in the treatment of chronic venous leg ulcers.

OBJECTIVE: Hypochlorous acid (HOCl) is a highly microbiocidal agent active against bacteria, viruses and fungi. Using quantitative microbiology, preliminary studies showed it achieved an appreciable reduction in the bacterial burden in chronic venous leg ulcers. The study aimed to determine whether it has a role as an additional treatment for chronic venous ulcers that have not healed with conventional treatment. METHOD: On the basis of previous reports we designed a study in which patients acted as their own controls, in that only patients who failed to achieve a 44% reduction in wound size with standard treatment (compression bandaging) received HOCl washes. RESULTS: Of 30 patients admitted to the study, 10 achieved a 44% ulcer reduction after three weeks of standard treatment. In addition to the standard compression treatment, the remaining 20 patients were given HOCl washes over 12 weeks. Of the 20 ulcers, nine (45%) healed and five (25%) reduced in size by over 60%. All patients became free of pain. CONCLUSION: These findings confirm the clinical efficacy of treating venous leg ulcers with hypochlorous washes. Use of HOCl washes as an adjunctive therapy for recalcitrant venous leg ulcers appreciably increases healing and rapidly relieves pain.

Aged↗
PubMed2004

Effect of glucose concentration on the growth of normal human dermal fibroblasts in vitro.

OBJECTIVE: Glucose requirements increase in tissue repair as glucose is an energy source for cell proliferation and the formation of extracellular matrix components. Glucose concentrations in leg ulcer wound fluid are lower than in normal human serum, with a median of 0.7 mM (range: 0.3-1.2 mM). This study investigated the effect of such low concentrations on the growth of fibroblasts in vitro. METHOD: Fibroblasts from 50-year-old and 87-year-old subjects were used. Growth in medium with various concentrations of glucose was determined by a colorimetric assay and microphotography. RESULTS: Up to day 6, there were minimal differences in growth, but after day 6 a clear dose-dependent increase in growth was observed. In the lower dose range (up to 2.3 mM), growth was highly dose-dependent (r2 = 0.981), with an increase of 1.8 mM stimulating cell growth by day 10 up to 163% of the controls (p < 0.0001). Concentrations of 5.5-25.5 mM glucose stimulated cell growth by day 10 to about 210% of the controls (p < 0.0001), with little difference between these concentrations. CONCLUSION: These results suggest that glucose enhancement to cells involved in healing might serve as an adjunctive treatment for chronic wounds.

Aged↗
PubMed2003

The cellular origins of the linear IgA disease target antigens: an indirect immunofluorescence study using cultured human keratinocytes and fibroblasts.

BACKGROUND: Linear IgA disease (LAD) is an IgA-mediated subepidermal immunobullous disease of adults and children, with heterogeneous immunopathology. Objectives To investigate to what extent the cellular origins of the target antigens account for the heterogeneity of the immune response in LAD. METHODS: Forty-nine adult and 33 childhood LAD sera were studied. Immunofluorescence was carried out to determine the expression of the LAD antigens by normal human keratinocytes, fibroblasts and mixed cultures of keratinocytes and fibroblasts. Immunoblotting was performed to determine the localization of the LAD target antigens in tissue extracts (48 adult and 31 childhood sera) and cell extracts (21 adult and 10 childhood sera). RESULTS: Thirty-one adult and 13 childhood LAD sera bound proteins expressed by human keratinocytes; of these sera, 15 adult and four childhood LAD sera also recognized proteins expressed by fibroblasts. A single adult serum was positive on fibroblasts alone. Seventeen adult and 20 childhood sera were negative on both cell types. There was a modest increase (9%) in the detection of the IgA autoantibodies on keratinocytes and fibroblasts grown together in mixed culture. Immunoblotting showed that the LAD target antigens could be detected in cell as well as in tissue extracts. CONCLUSIONS: Our results have shown that normal human keratinocytes and fibroblasts in culture express the LAD target antigens. LAD sera (with a single exception) bound antigens expressed by keratinocytes alone or by both keratinocytes and fibroblasts. The principal pattern of expression in keratinocytes was cytoplasmic, similar to that demonstrated by polyclonal antibodies to the 180-kDa bullous pemphigoid antigen (BP180). This reflects the pivotal role of BP180 in LAD. The finding that LAD antigens are expressed by both human keratinocytes and fibroblasts in culture may explain the heterogeneity of the target antigens, and may be a contributory factor in the immunopathology of the disease.

Adolescent↗
PubMed2003

Effect of intermittent radiant warming on proliferation of human dermal endothelial cells in vitro.

OBJECTIVE: This study investigated the effect of Warm-Up therapy on the proliferation of human microvascular dermal endothelial cells. METHOD: Endothelial cells from an adult subject were seeded in six-well plates and placed in an incubator at 32.5 degrees C. The following day Warm-Up dressings were placed over the plates, with or without warming cards. Cards set at 38 degrees C or 42 degrees C raised the temperature in the medium to maxima of 34.5 degrees C and 37.5 degrees C respectively. Units were switched on daily for three one-hour periods. Cell numbers were counted by haemocytometer. RESULTS: Maximum stimulation of endothelial cell proliferation occurred under the 38 degrees C card, with cells numbering 135-158% of the controls (p < 0.05). the 42 degrees C card also stimulated cells (110-155%) but this did not reach statistical significance. CONCLUSION: The accelerated proliferation of microvascular dermal endothelial cells achieved by intermittent radiant warming may have contributed to the increase in granulation tissue reported previously in our clinic.

Adult↗
PubMed2002

Effect of a lipopolysaccharide from E. coli on the proliferation of fibroblasts and keratinocytes in vitro.

Studies previously conducted in our laboratory have shown that an extract from the leaves of Chromo-laena odorata is mitogenic for human skin fibroblasts and keratinocytes. However, lipopolysaccharides, sometimes present in plant extracts, can also play a role in cell growth and might have been responsible for or contributed to the mitogenic activity observed. The present study aimed to investigate whether a lipopolysaccharide would have any effect on the proliferation of human fibroblasts and keratinocytes. Cells were seeded in 96-well plates and concentrations from 0.0 to 5.0 microg/mL of lipopolysaccharide in basal or growth medium were added. Cell growth was determined over a period of 10 days using a colorimetric assay. Lipopolysaccharide at concentrations between 0.05 microg/mL and 0.5 microg/mL in the growth medium significantly stimulated fibroblast proliferation after incubation for more than 6 days. In basal medium, more than 8 days of incubation was needed for significant stimulation of growth. Lipopolysaccharide stimulation of keratinocytes was evident at 0.5 microg/mL by day 3 in basal medium and by day 5 in growth medium. Although the lipopolysaccharide did stimulate cell growth it did so only at higher concentrations than were present in our plant extracts and to a lesser degree.

Cell Division↗
PubMed2002

Effect of Traumacel P on the growth of human dermal fibroblasts in vitro.

OBJECTIVE: It has been postulated that Traumacel P, a haemostatic powder, might assist the wound-healing process. This in vitro study investigated the effect of the powder on human dermal fibroblasts. METHOD: Experiments using fibroblasts from a 50-year-old donor were carried out using suspensions of Traumacel P ranging from 0.1 to 10.0 mg/ml. Dulbecco's Modified Eagle Medium with 10% or 0.4% foetal calf serum (FCS) and 5.5 mM or 25 mM glucose was used. The powder was either placed in contact with the cells or separated from them by a porous membrane. The response of the fibroblasts was assessed using the MTT assay. RESULTS: Concentrations of 0.5 mg/ml and 1.0 mg/ml stimulated the metabolic activity of the fibroblasts in both high and low glucose medium with 10% FCS, regardless of whether the powder was in contact with or separated from the cells. The greatest stimulation, to 174% of the controls, was produced by 0.5 mg/ml Traumacel P in low glucose/10% FCS with the powder in contact with the cells (p < 0.0001). Traumacel concentrations ranging from 0.5 to 5.0 mg/ml in 0.4% FCS significantly stimulated the metabolic activity of fibroblasts in low glucose medium, but not in the high glucose medium. CONCLUSION: These studies indicate that direct stimulation of fibroblast proliferation may be one of the ways in which Traumacel P aids the healing of ulcers observed in the clinic.

Cells, Cultured↗
PubMed2001

Identification and characterization of an Entamoeba histolytica upstream regulatory element 3 sequence-specific DNA-binding protein containing EF-hand motifs.

The hgl5 gene of Entamoeba histolytica is negatively regulated through the upstream regulatory element 3 (URE3) DNA motif TATTCTATT. This motif is also present and significant in the function of the E. histolytica fdx gene promoter. A yeast one-hybrid screen was used to identify an E. histolytica cDNA encoding a protein (URE3-BP) that recognized this DNA motif. Analysis of the predicted amino acid sequence demonstrated the presence of two EF-hand motifs but identified no canonical DNA binding motifs. URE3-BP, expressed in bacteria, demonstrated Ca(2+)-dependent and sequence-specific recognition of the URE3 DNA sequence as assessed by electrophoretic mobility shift assays. Antibodies raised against URE3-BP blocked the formation of the URE3 DNA-protein complex by native nuclear extracts. The URE3-BP protein was present in the E. histolytica nucleus and cytoplasm with an apparent molecular mass of 22.6 kDa. Our results represent the first use of a yeast genetic screen to identify, on the basis of function, a DNA-binding protein of an early branching eukaryote. Since the URE3 DNA can modulate gene expression in both a positive and negative manner, this protein may have more than one mechanism of interaction with transcriptional machinery. Characterization of URE3-BP should provide insight into transcription regulation and virulence control in this parasite.

Amino Acid Sequence↗
PubMed2001

Identification of essential active-site residues in the cyanogenic beta-glucosidase (linamarase) from cassava (Manihot esculenta Crantz) by site-directed mutagenesis.

The coding sequence of the mature cyanogenic beta-glucosidase (beta-glucoside glucohydrolase, EC 3.2.1.21; linamarase) was cloned into the vector pYX243 modified to contain the SUC2 yeast secretion signal sequence and expressed in Saccharomyces cerevisiae. The recombinant enzyme is active, glycosylated and showed similar stability to the plant protein. Michaelis constants for hydrolysis of the natural substrate, linamarin (K(m)=1.06 mM) and the synthetic p-nitrophenyl beta-D-glucopyranoside (PNP-Glc; K(m)=0.36 mM), as well as apparent pK(a) values of the free enzyme and the enzyme-substrate complexes (pK(E)(1)=4.4-4.8, pK(E)(2)=6.7-7.2, pK(ES)(1)=3.9-4.4, pK(ES)(2)=8.3) were very similar to those of the plant enzyme. Site-directed mutagenesis was carried out to study the function of active-site residues based on a homology model generated for the enzyme using the MODELLER program. Changing Glu-413 to Gly destroyed enzyme activity, consistent with it being the catalytic nucleophile. The Gln-339Glu mutation also abolished activity, confirming a function in positioning the catalytic diad. The Ala-201Val mutation shifted the pK(a) of the acid/base catalyst Glu-198 from 7.22 to 7.44, reflecting a change in its hydrophobic environment. A Phe-269Asn change increased K(m) for linamarin hydrolysis 16-fold (16.1 mM) and that for PNP-Glc only 2.5-fold (0.84 mM), demonstrating that Phe-269 contributes to the cyanogenic specificity of the cassava beta-glucosidase.

Binding Sites↗
PubMed2001

Identification of a novel low-temperature-response element in the promoter of the barley (Hordeum vulgare L) gene blt101.1.

Two winter barley (Hordeum vulgare L. cv. Igri) genomic clones, lambda gblt101.1 and lambda gblt101.2, encoding the blt101 gene family, were isolated from a genomic library. Deletion analysis of the blt101.1 promoter, using transient beta-glucuronidase (GUS) reporter expression assays, indicated that it contains at least three regulatory regions. A 107-bp region between nucleotides -168 and -275 with respect to the translation initiation codon, confers high-level GUS reporter expression at low temperature and contains a sequence (designated CR1) that is highly conserved in equivalent positions within the promoters of both members of the blt101 gene family. A 10-bp motif contained within CR1 binds proteins present in nuclear extracts from both control and low-temperature-treated barley tissue. Loss-of-function experiments, using transient-expression analysis, confirmed that this motif acts as a previously unreported low-temperature-responsive element. Nuclease sensitivity analysis of intact chromatin indicated that the blt101.1 promoter becomes more susceptible to DNase and micrococcal nuclease at low temperature, consistent with chromatin reorganisation upon transcriptional induction. It is proposed that both the 10-bp motif and chromatin reorganisation are involved in the regulation of blt101.1 at low temperature. This is the first detailed analysis of a low-temperature-specific plant promoter and identifies a novel low-temperature-response element.

Adaptation, Physiological↗
PubMed2001

Effects of Buddleja globosa leaf and its constituents relevant to wound healing.

An aqueous extract of Buddleja globosa leaves, used traditionally in Chile for wound healing, was tested for the ability to stimulate growth of fibroblasts in vitro and for antioxidant activity in the same fibroblast cell system challenged with hydrogen peroxide. Low concentrations of the extract gave an increase in fibroblast growth which was not statistically significant but cytotoxicity was observed at concentrations greater than 50 microg/ml. The extract showed strong antioxidant effect and fractionation led to the isolation of three flavonoids and two caffeic acid derivatives, each of which was shown to contribute to the antioxidant effect at concentrations below 10 microg/ml. These activities would accelerate the healing of wounds.

Antioxidants↗
PubMed2001

Intermediate subunit of the Gal/GalNAc lectin of Entamoeba histolytica is a member of a gene family containing multiple CXXC sequence motifs.

Killing by Entamoeba histolytica requires parasite adherence to host galactose- and N-acetyl-D-galactosamine (Gal/GalNAc)-containing cell surface receptors. A 260-kDa heterodimeric E. histolytica Gal/GalNAc lectin composed of heavy (Hgl) and light (Lgl) subunits has been previously described. Here we present the cloning and characterization of Igl, a 150-kDa intermediate subunit of the Gal/GalNAc lectin. Igl, Hgl, and Lgl colocalized on the surface membrane of trophozoites. Two unlinked copies of genes encoding Igl shared 81% amino acid sequence identity (GenBank accession no. AF337950 and AF337951). They encoded cysteine-rich proteins with amino- and carboxy-terminal hydrophobic signal sequences characteristic of glycosylphosphatidylinositol (GPI)-anchored membrane proteins. The igl genes lacked carbohydrate recognition domains but were members of a large family of amebic genes containing CXXC and CXC motifs. These data indicate that Igl is part of the parasite's multimolecular Gal/GalNAc adhesin required for host interaction.

Amino Acid Motifs↗
PubMed2001

Cloning and characterization of the pnb genes, encoding enzymes for 4-nitrobenzoate catabolism in Pseudomonas putida TW3.

Pseudomonas putida strain TW3 is able to metabolize 4-nitrotoluene via 4-nitrobenzoate (4NBen) and 3, 4-dihydroxybenzoic acid (protocatechuate [PCA]) to central metabolites. We have cloned, sequenced, and characterized a 6-kbp fragment of TW3 DNA which contains five genes, two of which encode the enzymes involved in the catabolism of 4NBen to PCA. In order, they encode a 4NBen reductase (PnbA) which is responsible for catalyzing the direct reduction of 4NBen to 4-hydroxylaminobenzoate with the oxidation of 2 mol of NADH per mol of 4NBen, a reductase-like enzyme (Orf1) which appears to have no function in the pathway, a regulator protein (PnbR) of the LysR family, a 4-hydroxylaminobenzoate lyase (PnbB) which catalyzes the conversion of 4-hydroxylaminobenzoate to PCA and ammonium, and a second lyase-like enzyme (Orf2) which is closely associated with pnbB but appears to have no function in the pathway. The central pnbR gene is transcribed in the opposite direction to the other four genes. These genes complete the characterization of the whole pathway of 4-nitrotoluene catabolism to the ring cleavage substrate PCA in P. putida strain TW3.

Ammonia-Lyases↗
PubMed2001

Emotion recognition by children with Down syndrome.

In three studies, children with Down syndrome were presented with emotion recognition tasks designed to tap their knowledge of simple emotions, their ability to label emotions, and their understanding of emotions from simple, story-based contexts. Results indicate that young children with Down syndrome perform similarly to typical controls matched on MAs of approximately 3 years. However, by developmental age of 4 years, children with Down syndrome performed worse than both MA-matched typical children and children with non-Down syndrome types of mental retardation. Although the MAs of children with Down syndrome increased over 2-years, their emotion recognition abilities did not. Taken together, findings suggest both etiological and developmental differences in the emotion recognition abilities of children with Down syndrome.

Affect↗
PubMed2000

Amebic liver abscess.

Amebiasis is a widespread parasitic disease caused by Entamoeba histolytica. This protozoan organism is the third leading parasitic cause of death in the developing world and is an important health risk to travelers in endemic areas. Amebiasis most commonly results in asymptomatic colonization of the gastrointestinal tract, but some patients may develop intestinal invasive disease or extraintestinal disease-amebic liver abscess being the most common extraintestinal manifestation. This article reviews epidemiologic features, pathophysiology, clinical features, diagnostic tests, imaging studies, treatment of amebic liver abscess, and prevention measures.

Animals↗
PubMed2000

Cloning and expression of ntnD, encoding a novel NAD(P)(+)-independent 4-nitrobenzyl alcohol dehydrogenase from Pseudomonas sp. Strain TW3.

Pseudomonas sp. strain TW3 is able to metabolize 4-nitrotoluene to 4-nitrobenzoate and toluene to benzoate aerobically via a route analogous to the upper pathway of the TOL plasmids. We report the cloning and characterization of a benzyl alcohol dehydrogenase gene (ntnD) which encodes the enzyme for the catabolism of 4-nitrobenzyl alcohol and benzyl alcohol to 4-nitrobenzaldehyde and benzaldehyde, respectively. The gene is located downstream of the previously reported ntn gene cluster. NtnD bears no similarity to the analogous TOL plasmid XylB (benzyl alcohol dehydrogenase) protein either in its biochemistry, being NAD(P)(+) independent and requiring assay via dye-linked electron transfer, or in its deduced amino acid sequence. It does, however, have significant similarity in its amino acid sequence to other NAD(P)(+)-independent alcohol dehydrogenases and contains signature patterns characteristic of type III flavin adenine dinucleotide-dependent alcohol oxidases. Reverse transcription-PCR demonstrated that ntnD is transcribed during growth on 4-nitrotoluene, although apparently not as part of the same transcript as the other ntn genes. The substrate specificity of the enzyme expressed from the cloned and overexpressed gene was similar to the activity expressed from strain TW3 grown on 4-nitrotoluene, providing evidence that ntnD is the previously unidentified gene in the pathway of 4-nitrotoluene catabolism. Examination of the 14.8-kb region around the ntn genes suggests that one or more recombination events have been involved in the formation of their current organization.

Alcohol Oxidoreductases↗
PubMed2000

Investigating plant-based medicines for wound healing with the use of cell culture technologies and in vitro models: a review.

INTRODUCTION: Cell culture and molecular technologies are basic yet sophisticated research tool used to investigate plant-based medicine for wound healing. METHODS: Cell viability and proliferation assay is used to determine whether there are any positive effects and to discover what is the limiting cytotoxic concentration in vitro. The scratch technique, fibroblast-populated collagen lattices and aortic rings embedded gels are used as the in-vitro models of wound re-epithelialization, contraction and angiogenesis. The immunofluorescence, immunoblotting and organotypic culture can be used to detect expression of specific proteins that are modulated by plant extracts during the wound healing process. MAIN FINDINGS: Given the dynamics of the wound healing process, cell culture and molecular technologies are advantageous in providing us with detailed studies and analysis of each intricate process. CONCLUSION: The scientific approaches for the study of traditional plant-based remedies for wound healing will provide us an important platform for rigorous testing and evaluation of their clinical efficacy based on accepted rules of evidence.

Cell Survival↗
PubMed1999

Enhanced ultrasensitive detection of structurally diverse antigens using a single immuno-PCR assay protocol.

Our studies of DNA damage and repair in autoimmune disease, lymphomagenesis, and carcinogenesis, require identification of an immunoassay approach that is capable of ultrasensitive detection in a routine human tissue biopsy of several physicochemically diverse antigens, some of which will be present at very low level. Immuno-polymerase chain reaction (immuno-PCR) is a recently described method for ultrasensitive antigen detection that combines the amplification power of PCR with a method similar to a standard antibody capture, enzyme-linked immunosorbent assay (ELISA). As a test of the universality of immuno-PCR, and as an assessment of the suitability of this method for our studies, we used a single immuno-PCR protocol to assay purified forms of the following physicochemically diverse antigens: oligomeric pyruvate dehydrogenase complex (PDC; Mr 8.5 x 10(6)), the promutagenic DNA base adduct O(6)-methylguanosine (Mr 298) and its monomeric repair enzyme, O(6)-methylguanine-DNA methyltransferase (MGMT; Mr 22,000), and a peptide from the N-terminus of MGMT (Mr 2310). We found that all antigens could be ultrasensitively assayed using the single immuno-PCR protocol. Assay limits observed using antigen-specific (primary) antibodies at 1 microg/ml, were in the approximate range of 10(2)-10(9) molecules, with O(6)-methylguanosine being detected most sensitively. Sensitivity of the antigen assay appeared to positively correlate with primary antibody titres determined by ELISA. Furthermore, we observed a substantial increase in detection sensitivity for all antigens by the use of primary antibodies at the higher level of 10 microg/ml. The latter approach permitted antigen assay within the approximate range of 10(0)-10(7) molecules. The combination of higher titre primary antibodies and their use at higher input level, produced an increase of immuno-PCR assay sensitivity of up to four orders of magnitude greater than those previously reported through the use of this assay to measure other antigens. This represents up to a nine order of magnitude increase in immunoassay sensitivity compared to ELISA. Our findings provide compelling evidence that immuno-PCR is indeed a universal ultrasensitive antigen detection method. Using the indicated assay enhancements. immuno-PCR performed as detailed here can offer greatly increased sensitivity for antigen measurement compared to other methods. Thus, our findings suggest that parallel quantitation of several different antigens in very small samples of human tissue will be readily attainable using immuno-PCR.

Amino Acid Sequence↗