Biomedical subjects

S B Brown

Publications and source records attributed to S B Brown.

At least 55 records · Page 3Linked to original sources

PubMed1998

The induction of apoptosis by a positively charged methylene blue derivative.

Identifying the cellular responses to photodynamic therapy (PDT) is important if the mechanisms of cell death are to be fully understood. PDT with a methylene blue analog DO15 yielded mitochondrial photodamage whilst membrane and lysosomal integrity were maintained. Apoptosis was detected using the DNA stain HO342, by the appearance of 50 kb fragments and by DNA ladder formation. The release of mono- and oligonucleosomes was further quantified using an ELISA protocol. Large DNA fragments were observed immediately following illumination, and nucleosomes were detected at 1-2 h post-treatment. Increasing the dose 4-fold accelerated the apoptotic response to PDT. This is the first report of a thiazine photosensitiser inducing apoptosis and is consistent with recent proposals suggesting that release of mitochondrial components may play an important role in the mechanism of cell death.

Animals↗

PubMed1998

The photodynamic response of two rodent tumour models to four zinc (II)-substituted phthalocyanines.

Four novel zinc (II)-substituted phthalocyanines, varying in charge and hydrophobicity, were evaluated in vivo as new photosensitizers for photodynamic therapy. Two rat tumours with differing vascularity were used: a mammary carcinoma (LMC1) and a fibrosarcoma (LSBD1), with vascular components six times higher in the latter (10.8%+/-1.5) than in the former (1.8%+/-1.4). Each sensitizer was assessed for tumour response relative to normal tissue damage, and optimum doses were selected for further study, ranging from 0.5 to 20 mg kg(-1). Interstitial illumination of the tumours was carried out using a 200-microm-core optical fibre with a 0.5 cm length of diffusing tip, at either 680 or 692 nm, depending on the sensitizer. Light doses of between 200 and 600 J were delivered at a rate of 100 mW from the 0.5-cm diffusing section of the fibre. Maximum mean growth delays ranged from 9 to 13.5 days depending on sensitizer and type of tumour, with the most potent photosensitizer appearing to be the cationic compound. Histopathological changes were investigated after treatment to determine the mechanism by which tumour necrosis was effected. The tumours had the appearance of an infarct and, under the conditions used, the observed damage was shown to be mainly due to ischaemic processes, although some direct tumour cell damage could not be ruled out.

Animals↗

PubMed1998

Maternal blood, egg and larval thiamin levels correlate with larval survival in landlocked Atlantic salmon.

A link was previously established between the Cayuga syndrome, a condition causing 100% mortality in larval landlocked Atlantic salmon, Salmo salar, in several of New York's Finger Lakes, and a maternal diet of alewife, Alosa pseudoharengus, a non-native thiaminase-rich Clupeid fish. We evaluated salmon larvae viability relative to maternal thiamin status, and investigated the putative link of the Cayuga syndrome to an alewife diet in fish from the geographic regions outside the Finger Lakes/lower Great Lakes watersheds. We identified Cayuga syndrome in Atlantic salmon from Otsego Lake in the Susquehanna River watershed and from Green Pond in New York's Adirondack Mountains. In both systems alewife represent the major component of the diet for the salmon. Thiamin levels in the maternal blood of Otsego salmon with syndrome-negative progeny were three- to four-fold greater than those Otsego females whose progeny exhibited 100% mortality. Thiamin levels in eggs and larvae were directly related to thiamin levels in maternal blood in both syndrome-positive and syndrome-negative stocks. Thiamin bath treatments of syndrome-afflicted larvae eliminated mortality regardless of their lake stock of origin. Maternal blood levels of approximately 0.31 nmol thiamin pyrophosphate/g or 0.44 nmol total thiamin/g appear necessary to achieve egg threshold levels of approximately 0.8 and 1.1 nmol/g unphosphorylated and total thiamin, respectively; these egg thiamin levels should prevent significant syndrome-related mortality in landlocked Atlantic salmon larvae. These results confirm the role of thiamin in the etiology of the Cayuga syndrome and support the dietary link of this naturally occurring thiamin deficiency to the thiaminase-rich alewife.

Animals↗

PubMed1998

Fluorescence photobleaching of ALA-induced protoporphyrin IX during photodynamic therapy of normal hairless mouse skin: the effect of light dose and irradiance and the resulting biological effect.

The photobleaching of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) was investigated during superficial photodynamic therapy (PDT) in normal skin of the SKH HR1 hairless mouse. The effects of light dose and fluence rate on the dynamics and magnitude of photobleaching and on the corresponding PDT-induced damage were examined. The results show that the PDT damage cannot be predicted by the total light dose. Photobleaching was monitored over a wide range of initial PpIX fluorescence intensities. The rate of PpIX photobleaching is not a simple function of fluence rate but is dependent on the initial concentration of sensitizer. Also, at high fluence rates (50-150 mW/cm2, 514 nm) oxygen depletion is shown to have a significant effect. The rate of photobleaching with respect to light dose and the corresponding PDT damage both increase with decreasing fluence rate. We therefore suggest that the definition of a bleaching dose as the light dose that causes a 1/e reduction in fluorescence signal is insufficient to describe the dynamics of photobleaching and PDT-induced damage. We have detected the formation of PpIX photoproducts during the initial period of irradiation that were themselves subsequently photobleached. In the absence of oxygen, PpIX and its photoproducts are not photobleached. We present a method of calculating a therapeutic dose delivered during superficial PDT that demonstrates a strong correlation with PDT damage.

Aminolevulinic Acid↗

PubMed1997

Characterization and localization of mitochondrial oligopeptidase (MOP) (EC 3.4.24.16) activity in the human cervical adenocarcinoma cell line HeLa.

In this study we describe the partial purification and characterization of the HeLa cell oligopeptidase M or endopeptidase 3.4.24.16. The HeLa enzyme was isolated initially by its ability to hydrolyse a nonapeptide substrate (P9) which was cognate to the N-terminal cleavage site of preproTGF alpha. The enzyme was shown to be a metalloprotease as it was inhibited by Zn(2+)-chelating agents and DTT, and had an approximate molecular weight of 55-63 kD determined by gel filtration. Neurotensin, dynorphin A1-17 and GnRH1-9 were rapidly degraded by the enzyme while GnRH1-10 and somatostatin were not. Neurotensin was cleaved at the Pro10-Tyr11 bond, leading to the formation of neurotensin (1-10) and neurotensin (11-13). The K(m) for neurotensin cleavage was 7 microM and the Ki for the specific 24.16 dipeptide inhibitor (Pro-ile) was 140 microM which were similar to those observed from the human brain enzyme [Vincent et al. (1996): Brain Res 709:51-58]. Through the use of specific antibodies, the purified HeLa enzyme was shown to be oligopeptidase M. This enzyme and its closely related family member thimet oligopeptidase were shown to co-elute during the isolation procedure but were finally separated using a MonoQ column. Oligopeptidase M is located mainly in mitochondria though it was detected on the plasma membrane in an inactive form. The results obtained demonstrate the first recorded instance of this enzyme in human tissue cultured cells, and raise the issue of its function therein.

Cell Membrane↗

PubMed1997

Actin is cleaved during constitutive apoptosis.

Proteases play an important role in the programme of cell death by apoptosis but little is known of the substrates cleaved, particularly in constitutive models of this type of cell death. Neutrophils spontaneously undergo apoptosis in culture without requiring external stimuli. During this process we found biochemical and immunochemical evidence for the cleavage of membrane-associated actin, a component of the cytoskeleton that links polymerized actin to the plasma membrane. Cleavage occurred at a single site at the N-terminus, between residues Val43-Met44, a site devoid of a consensus motif for cleavage by cysteine proteases of the interleukin-1beta-converting enzyme (ICE)-family. Whereas actin cleavage and nuclear/cell surface markers of apoptosis were co-ordinately diminished by zVAD-fmk, an inhibitor of the ICE-like family of proteases, only acetyl-leucyl-leucylnormethional, an inhibitor of calpains, was capable of completely inhibiting actin cleavage. Our results suggest that actin is not a direct substrate for the ICE-like family of proteases. By disabling the cytoskeleton, actin cleavage may be an important component in the capacity of apoptosis to reduce the injurious potential of neutrophils.

Actins↗

PubMed1997

"Spot 14" protein functions at the pretranslational level in the regulation of hepatic metabolism by thyroid hormone and glucose.

"Spot 14" protein appears rapidly in nuclei of hepatocytes exposed to glucose and thyroid hormone. Exposure of glucose- and T3-treated hepatocytes to a spot 14 antisense oligonucleotide inhibited induction of mRNAs encoding malic enzyme, ATP citrate-lyase, fatty acid synthase, liver-type pyruvate kinase, phosphoenolpyruvate carboxykinase, and type I deiodinase but not hydroxymethylglutaryl-CoA reductase, cytochrome c, and actin mRNAs. Induction of spot 14, ATP citrate-lyase, and fatty acid synthase polypeptides, but not propionyl-CoA carboxylase and mitochondrial pyruvate carboxylase, was inhibited. Antisense treatment of hepatocytes transfected with a reporter controlled by a glucose- and T3-inducible fragment of the pyruvate kinase gene promoter inhibited reporter activity, as did cotransfection of the reporter and a spot 14 antisense plasmid. Spot 14 protein acts in the induction of mRNAs coding for key lipogenic (malic enzyme, ATP citrate-lyase, fatty acid synthase), glycolytic (pyruvate kinase), and gluconeogenic enzymes (phosphoenolpyruvate carboxykinase), as well as the diet-responsive type I deiodinase, but not those involved in mitochondrial respiration (cytochrome c) or cholesterol synthesis (hydroxymethylglutaryl-CoA reductase). Transfection experiments indicated that these effects are mediated at the transcriptional level. The protein functions in the activation of genes involved in metabolic switching between the fasted and fed states in liver.

ATP Citrate (pro-S)-Lyase↗

PubMed1997

Purification and identification of apophycocyanin alpha and beta subunits from soluble protein extracts of the red alga Cyanidium caldarium. Light exposure is not a prerequisite for biosynthesis of the protein moiety of this photosynthetic accessory pigment.

Much controversy exists as to the level at which light exerts control over the biosynthesis of the photosynthetic apparatus in higher plants and other organisms. The eukaryotic red alga Cyanidium caldarium, like higher plants, undergoes light induction of chlorophyll synthesis. In addition to chlorophyll a the alga also synthesises the linear tetrapyrrole phycocyanobilin, which is combined with alpha or beta apobiliproteins to form phycocyanin, the major light-harvesting pigment in this organism. We have previously shown that the tetrapyrrole precursor 5-aminolaevulinic acid (ALA) can substitute for light in inducing the biosynthesis of the phycocyanobilin moiety of this protein. We have also described the appearance of a protein of similar isoelectric point and molecular weight to phycocyanin in ALA-fed cells (Turner et al., 1992, Plant Physiol Biochem 30: 309-314). We now report on the protein's immunological and sequence identity with phycocyanin alpha and beta subunits, and provide further evidence that bilin-apoprotein ligation is light dependent.

Amino Acid Sequence↗

PubMed1997

The subcellular localization of Zn(II) phthalocyanines and their redistribution on exposure to light.

The development of second-generation photosensitizers to improve photodynamic therapy (PDT) is an area of extensive research. Three such compounds that have been synthesized in our group are polysubstituted Zn(II) phthalocyanines that differ in their overall net charge (one cationic, one anionic and one neutral). The aim of this study was to characterize the drugs in terms of their uptake, cell killing efficacy and subcellular localization in RIF-1 cells in vitro and to identify any possible structure/function relationships. The results show that the relative uptakes and cell killing efficacy of each of the drugs follows the order cationic > > neutral > anionic. For the anionic and cationic drugs the initial subcellular localization was in the lysosomes as determined by fluorescence microscopy. The neutral phthalocyanine demonstrated a more diffuse localization characteristic of membrane staining with some involvement of the Golgi apparatus in the perinuclear area. Following light exposure the drugs rapidly relocalized to different sites within the cell in a manner that was apparently charge dependent and this relocalization was accompanied by an increase in the fluorescence associated with the drugs. This indicates that the primary sites of localization of these photosensitizers may not be as important as their secondary sites in producing cell killing during PDT, especially as the fluorescence intensity increases on relocalization.

Animals↗

PubMed1997

Surface treatment effects on amalgam repair strength.

PURPOSE: To determine whether the use of microetching and bonding agents could improve the shear bond strength between aged and freshly added dental amalgam, simulating an amalgam repair. MATERIALS AND METHODS: Aged amalgam surfaces were treated with either a carbide bur or carbide bur + microetcher (Al2O3 abrasive). Five commercially-available adhesive systems were tested. Fresh amalgam was condensed through a split mold onto each prepared amalgam surface. Two control groups were included: a positive control consisting of non-repaired amalgam, and a negative control consisting of aged specimens which received neither carbide bur nor microetching preparation prior to the addition of fresh amalgam. In addition, one group received only carbide bur and another received only carbide bur and microetching treatments, without any adhesive. Following storage and thermocycling, each specimen was tested for shear bond strength. RESULTS: The mean shear bond strength of the repaired samples treated with carbide bur alone (25.8 MPa) was not exceeded by any of the other treatments. Neither microetching nor use of an adhesive agent significantly improved amalgam-to-amalgam shear bond strength. In some cases, use of an adhesive agent significantly decreased the shear bond strength of repaired amalgam samples.

Aluminum Oxide↗

PubMed1996

Loss and shedding of surface markers from the leukemic myeloid monocytic line THP-1 induced to undergo apoptosis.

Previous studies have established that a relationship exists between apoptosis and cell surface (ecto-) peptidase activity. Thus dose-dependent increases were found both in ectopeptidase activities and in the proportion of cells undergoing apoptosis in HeLa cell monolayers after exposure to UV and other perturbants causing arrest of DNA synthesis (indirectly or directly as a result of DNA damage). The nature of the correlation made no distinction as to whether an increase in peptidase activity was causal of, or consequential to apoptosis, nor whether the increase was a general response by all cells. As a wider approach to understanding the possible role played by ectopeptidases in apoptosis, we report the effect on expression of a known ectopeptidase, aminopeptidase N (CD13), by a myelomonocytic cell line induced to undergo apoptosis. Using THP-1 cultures exposed to low concentrations of ethanol, we used FACS technology to sort for early apoptotic cells that have an increased ability to sequester the vital dye Hoechst 33342 while excluding nonvital dyes. Apoptosis was verified by light, fluorescence, and transmission electron microscopy, and the presence of DNA fragmentation. These early apoptotic cells showed a significant loss in CD13 labeling. Another surface marker, CD33, behaved similarly, whereas CD14 was lost globally, and not just by the apoptotic cells. Peptidase assays confirmed that an aminopeptidase was shed into the bathing media and that this activity was inhibitable both by bestatin and by a CD13 neutralizing monoclonal antibody. In treated cells, there was no evidence for an increase in cell surface protease activity directed toward a highly aliphatic nonapeptide substrate used as a model for TGF-alpha scission from its precursor form. However, other cell surface proteases of different specificity are presumably responsible for the observed shedding of CD13.

Amino Acid Sequence↗

PubMed1996

Kinetic analysis of thyroid hormone secretion and interconversion in the 5-day-fasted rainbow trout, Oncorhynchus mykiss.

Estimating the 3,5,3'-triiodothyronine (T3) thyroidal secretion rate and the rate of extrathyroidal conversion of thyroxine (T4) to T3 are two difficult and important quantitative endocrine system problems in vertebrates. To address these questions in fish, two thyroid hormone tracer studies were modeled and analyzed, based on data from two groups of rainbow trout maintained at 12 degrees and fasted for 5 days. The data consisted of three time series: plasma concentrations of radioactive 125I-labeled T3 (T3*) following T3* injection, and both labeled T4 (T4*) and T3* following T4* injection. To facilitate model parameter estimation, plasma volumes were determined independently by injection of labeled bovine serum albumin. The T4* injectate was contaminated by an unknown amount of T3*, and this was considered an additional unknown. A six-compartment model was formulated in terms of 13 uniquely identifiable (quantifiable) parameters, which were estimated simultaneously from the three data sets using a sophisticated optimization algorithm built into a new model-fitting software package called FITMOD. The rates of interest, plus other kinetic indices, were estimated successfully using additional analysis. We found that the thyroid gland secreted 0.835 +/- 0.707 (mean +/- SD) pmol/hr of T3 and 2.44 +/- 2.09 pmol/hr of T4 per 100 g body weight (BW). Also, 8.19 to 11.2% of secreted T4 was monodeiodinated to T3, forming 0.200 to 0.274 pmol/hr of T3 per 100 g BW. This means that 75 to 81% of all T3 produced was secreted by the thyroid in these starved fish--a rather surprising result--while the remaining 19 to 25% resulted from T4 to T3 conversion.

Algorithms↗

PubMed1995

Photosensitization of the endometrium with topical 5-aminolevulinic acid.

Photosensitization of the endometrium was attempted in vitro and in vivo by intrauterine administration of 5-aminolevulinic acid, which is converted to the photosensitizer protoporphyrin IX. Protoporphyrin IX was found in both functional and basal layers of the endometrial glands at concentrations nine and 10 times higher than myometrium in in vitro and in vivo experiments, respectively. Selective endometrial photosensitization is possible with topical 5-aminolevulinic acid, but it might not be distributed sufficiently evenly for use as a sensitizing agent in photodynamic ablation.

Administration, Topical↗

PubMed1995

The quantitative determination of Photofrin and Polyhaematoporphyrin in plasma: pitfalls and inaccuracies.

An accurate and relatively rapid fluorometric assay for Photofrin, or our own preparation Polyhaematoporphyrin (PHP), in plasma has been developed. This method takes into account the significant proportion of hydrolysis-resistant material now known to be present in these sensitizers, which has undoubtedly led to the inaccurate assessment of these drugs and other preparations of haematoporphyrin derivative (HPD) in numerous studies. The method was devised to allow for incomplete hydrolysis by calibration with plasma samples to which known amounts of photosensitizer were added in vitro. It was then compared with an "absolute" method in which plasma from animals that had received 14C-labelled drug was subjected to radioactivity assay. The two approaches gave almost identical results. The calibration method is applicable to the determination of any HPD-derived drug from patient or animal studies. As an example of its use in the present study, it was applied to the determination of the pharmacokinetics of PHP in the rat.

Animals↗

PubMed1995

Response of a rodent fibrosarcoma to photodynamic therapy using 5-aminolaevulinic acid or polyhaematoporphyrin.

The effects of 5-aminolaevulinic acid (ALA)-based photodynamic therapy (PDT) on the growth of an intradermal rat fibrosarcoma have been determined following topical, intravenous, or intratumour application of ALA. The pattern of tumour growth was identical following each type of therapy and we conclude that the efficacy of ALA-PDT is independent of the route of drug administration, at least in this tumour system. We have also compared the efficacy of interstitial ALA-based PDT with similar therapy using a conventional photosensitizer, polyhaematoporphyrin (PHP). ALA-PDT was less effective than PHP-based therapy at inhibiting the growth of a subcutaneous rat fibrosarcoma at light doses over 300 J. It is suggested that protoporphyrin IX photo-bleaching may limit the effectiveness of treatment, whilst the more bleach-resistant PHP may continue to be effective at higher light doses.

Aminolevulinic Acid↗

PubMed1994

Induction of cell surface peptidase activity: a global response to cell stress correlated with apoptosis.

We have previously characterized the stimulation of HeLa cell surface peptidase activity directed toward a nonapeptide substrate in response to low fluences of ultraviolet irradiation [Brown et al. (1993): J Cell Biochem 51:102-115]. To explore the hypothesis that this comprised a global response to cell stress featuring the interruption of DNA synthesis, a variety of agents affecting macromolecular synthesis were applied to HeLa cell cultures. Actinomycin D, 5,6-dichloro-1 beta-ribofuranosyl benzimadazole, mitomycin C, ultraviolet light, and cycloheximide at doses which inhibited cell growth, but fell short of increasing the proportion of cells which had lost cell membrane impermeability to trypan blue, resulted in the concentration dependent increase in both amino- and endo-peptidase activities of intact HeLa cell cultures. gamma-Irradiation, despite inhibiting an increase in cell number over a 20-h observation period, had no effect on the expressed level of cell surface peptidase activity nor did the accumulation of cells in S or G2 phase by thymidine parasynchronization. Some of these agents were found to increase the proportion of cells in the culture undergoing apoptosis (programmed cell death), and a strong correlation was found between the extent of apoptosis and the degree of elevation in cell surface peptidase activity. Higher concentrations of perturbants in some instances increased the percentage of cells that were nonviable and an associated release of intracellular proteases overwhelmed the linear correlation with apoptotic cells. The present data do not distinguish between a homogeneous elevation of surface peptidase activity in all cells of treated cultures or the heterogeneous increase in only preapoptotic or apoptotic cells. Since sunburn of the skin increases both the occurrence of apoptotic keratinocytes (sunburn cells) in the affected epidermis and the release of membrane bound cell activators such as transforming growth factor alpha, it is suggested by way of extrapolation of these in vitro results, that the increase in cell surface proteolytic activity plays an integral part in the reparative responses of the epidermal cells in vivo.

Amino Acid Sequence↗

PubMed1994

Coprolalia in younger patients with Gilles de la Tourette syndrome.

The objective of this study was to determine the frequency of coprolalia in younger patients with Gilles de la Tourette syndrome (GTS). Coprolalia, which is the least understood and perhaps most unusual symptom of GTS, is reported to occur in 4-60% of all patients with GTS. Most reports indicate a prevalence > 30%. The widely varying prevalence figures for coprolalia may reflect cultural differences, severity of disease, and the age of the population surveyed. This study is a chart review of 112 patients seen in a pediatric neurology clinic in South Florida who met the Diagnostic and Statistical Manual of Mental Disorder (3rd ed., revised) criteria for GTS. Only 8% of the patients in the study exhibited coprolalia. This unusually low frequency of coprolalia suggests that coprolalia may be rarer than previously believed in younger patients. It may also better reflect the frequency of coprolalia in a community-based pediatric neurology practice.

Adolescent↗

PubMed1994

On the photodynamic therapy action spectrum of zinc phthalocyanine tetrasulphonic acid in vivo.

The photodynamic therapy (PDT) activity of zinc phthalocyanine tetrasulphonic acid in a rodent tumour model was shown to be critically dependent on the wavelength of the excitation laser light over a relatively small wavelength range. Thus the sensitizer showed a doubling of the PDT activity with fibrosarcoma LSBD1 in BDIX rats when the wavelength of the illuminant was displaced from 680 to 692 nm. Under these conditions, the sensitizer is approximately three times more effective than polyhaematoporphyrin, whereas previously it has been considered to be of low PDT activity. This wavelength effect is attributed to a red shift of the absorption spectrum of the sensitizer in cells compared with that in solution. Fluorescence excitation studies with sensitizer absorbed in mouse 3T3 fibroblast cells are consistent with such a red shift.

Animals↗