Biomedical subjects

M J Groves

Publications and source records attributed to M J Groves.

At least 19 recordsLinked to original sources

PubMed2001

Effect of FITC-dextran molecular weight on its release from floating cetyl alcohol and HPMC tablets.

The release mechanism of high molecular weight fluorescein isothiocyanate dextrans (FITC-dextrans) from HPMC hydrogel matrices was studied. An anomaly was noted in the release behaviour of a series of high molecular weight FITC-dextrans from a tablet formulation designed to float in stomach contents. The tablets contained sodium bicarbonate and hydroxypropylmethyl cellulose (HPMC) in a cetyl alcohol matrix. When hydrated in an acid medium, this tablet consisted of a mixed solid with a viscous surface layer containing carbon dioxide bubbles through which the active ingredient (FITC-dextran) was released into the aqueous environment. However, it was observed that, above a critical molecular weight (approx. 65 kDa), the FITC-dextran was only released into the medium by an erosion-type mechanism, whereas, below this value, both diffusion and erosion processes took place. The key constraint appeared to be the apparent gel pore-size of the hydrated HPMC that was approximately 12 nm in diameter, irrespective of the molecular weight of the HPMC samples evaluated. It was concluded that FITC-dextran release was controlled by both FITC-dextran molecular weight and the HPMC hydrogel structure.

Dextrans↗

PubMed2001

Acute cardio-respiratory effects in rats of PS4alpha, an antineoplastic peptidoglycan from Mycobacterium vaccae.

PS4alpha is a high molecular weight peptidoglycan extracted from Mycobacterium vaccae, which has demonstrated considerable antineoplastic activity in-vivo without apparent toxicity. Available fortesting in only small quantities, a sensitive in-vivo method for measuring pulse and breathing rates in cannulated rats was applied to this compound at doses of 5, 50 and 500 microg kg(-1). Various parameters (mean arterial pressure, maximum transpulmonary pressure, compliance, heart rate, minute volume, respiratory rate and tidal volume) were followed for up to 1 h and demonstrated no significant deviation in the baseline values obtained before injection. This compound at doses up to 500 microg kg(-1) had no apparent acute toxicity in rats, but chronic effects at this and higher doses have to be determined by more conventional toxicological methods before proceeding to evaluate PS4alpha as an antineoplastic agent.

Animals↗

PubMed2000

Isolation and identification of poly-alpha-(1-->4)-linked 3-O-methyl-D-mannopyranose from a hot-water extract of Mycobacterium vaccae.

A polysaccharide around 3.6 kDa has been identified as the major carbohydrate moiety of a antineoplastic protein-polysaccharide complex (PS4A) obtained by boiling intact cells of Mycobacterium vaccae in water. 1H and 13C NMR spectra of this polysaccharide suggested it was a highly homogeneous polymer composed substantially of one monomer, probably an alpha-linked O-methylated mannose. Comparison of the COSY spectra of the original and acetylated polymer indicated that the glycosidic linkage and the methyl ether were interchangeable, at O-3 and O-4. Further study demonstrated that the benzyolated hydrolysate of the polymer was 1,2,4,6-tetra-O-benzoyl-3-O-methyl-beta-mannopyranose. The hydrolysate was 3-O-methyl-alpha, beta-mannopyranose and the polymer was therefore poly-alpha-(1-->4)-linked 3-O-methyl-D-mannopyranose. This conclusion was further confirmed with an authentic sample of the monomer, which had spectral data identical to those of the hydrolyzate and co-eluted from an ion-exchange HPLC with the major sugar in the hydrolysate.

Adjuvants, Immunologic↗

PubMed2000

Glutathione S-transferase enzyme expression in hematopoietic cell lines implies a differential protective role for T1 and A1 isoenzymes in erythroid and for M1 in lymphoid lineages.

BACKGROUND AND OBJECTIVES: Glutathione S-transferases (GSTs) are phase II metabolizing enzymes which catalyze the conjugation of glutathione (GSH) to electrophilic substrates and possess selenium-independent glutathione peroxidase activity. The GST enzyme family includes the cytosolic isoforms GST-alpha, mu (GSTM), pi (GSTP), theta (GSTT) and sigma (GSTS). GSTT1, P1 and M1 are polymorphic and altered polymorphic frequency of genes encoding these proteins has been suggested as a potential risk factor for the development of hematopoietic malignancies. Overexpression of GSTs has also been implicated in chemotherapeutic drug resistance. This study was undertaken to elucidate the potential functional relevance of these genetic polymorphisms in hematopoiesis. DESIGN AND METHODS: GST genotype of 14 hematopoietic cell lines was determined by polymerase- chain-reaction (PCR). Gene expression of GSTs in a cell line was detected by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) on TaqMan 7700 and by semi-quantitative RT-PCR. Cytosolic GST protein expression was detected by Western blot. GST conjugation activity was assayed using 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. RESULTS: GSTP1 expression was higher than other GSTs in 13/14 cell lines and paralleled CDNB conjugation activity. GSTP1 and GSTM1 predominated in lymphoid lines whilst T1 expression was relatively greatest in erythroid lines but was absent in 7/12 non-null lines. GSTT2 was expressed in only 3/4 lines. The 3 cell lines which expressed GSTA1 were all erythroid. INTERPRETATION AND CONCLUSIONS: Glutathione S-transerases showed differential lineage expression in hematopoietic cell lines. This implies a greater cytoprotective role for GSTT1 and GSTA1 in erythroid cells and GSTM1 in lymphoid cells. We postulate that inherited gene deletion of GSTT1 and M1 may produce increased genotoxic susceptibility for erythroid and lymphoid cell respectively, following exposure to xenobiotics that are substrates for these enzymes.

Cell Lineage↗

PubMed1999

Inhibition of sensory neuron apoptosis and prevention of loss by NT-3 administration following axotomy.

Following permanent transection of their peripheral axons, a proportion of adult rat dorsal root ganglion neurons undergo programmed cell death (apoptosis) over a period of months. The underlying causes of this neuron loss are unclear, but may involve the interruption of the supply of target-derived neurotrophic factors, the replacement of which could prevent this loss from occurring. To investigate whether the administration of neurotrophic factors can prevent the dorsal root ganglion neuron death in adults, a 1 mg/ml solution of ciliary neurotrophic factor or of NT-3 was applied via a silicon reservoir to the proximal stump after unilateral sciatic transection at mid-thigh level. The incidence of apoptotic neurons and neuronal loss in the L4 and L5 ganglia ipsilateral to sciatic nerve transection when compared with the contralateral ganglia was then measured 1 month later. This was assessed by examining serial sections of ganglia for neurons undergoing apoptosis and expressing the total counted as a percentage of the total number of neurons estimated using a stereological neuron counting technique. Our results show that NT-3 administration significantly reduced the incidence of apoptotic neurons and prevented neuron loss, while CNTF had no effect on either parameter.

Animals↗

PubMed1999

Expression of three oligosaccharide conjugates by neonatal rat dorsal root ganglion neurons: comparison with CGRP and GAP43 immunoreactivity.

Adult dorsal root ganglion neurons express oligosaccharides conjugated to lipids that may be involved in cell-cell recognition, and consequently in the laminar organisation of their central terminations. This paper describes an immunohistochemical study of the developmental expression of 2 lactoseries (LA4 and LD2) and 1 globoseries (SSEA4) oligosaccharide conjugates in rats from embryonic d 19 to postnatal d 60. The expression of calcitonin gene related peptide and the growth associated protein GAP43 was also examined for comparative purposes. We found that these oligosaccharide conjugates begin to be expressed after birth, suggesting that they may be involved in maturation of the central or peripheral terminations, rather than axonal guidance.

Animals↗

PubMed1999

Formulation and biological activity of antineoplastic proteoglycans derived from Mycobacterium vaccae in chitosan nanoparticles.

Although heat-killed suspensions of Mycobacterium vaccae have been tested clinically against tuberculosis and cancer, from a pharmaceutical perspective it would be advantageous to utilize isolated active components rather than the heat-degraded bacterial materials. In our laboratory we have isolated from M. vaccae a number of high-molecular-weight proteoglycans with considerable immunological and antineoplastic activity. The structure of one of these, PS4A, obtained by extraction with boiling water, seems to consist of a basic unit with a 20-kDa protein core to which are attached glucans and O-methylated 4-kDa polysaccharides. The molecular weight is (approx.) 50 kDa, but because of self-association, that of the recovered high-molecular-weight fraction is greater than 150 kDa. A similar, but even larger, molecule (PS4alpha, MW approximately 20 MDa) is obtained by cold extraction with 8 M urea. Both are active in-vivo against an S-180 murine sarcoma model but have no activity in-vitro, suggesting an antitumour effect involving activated macrophages. For this reason gelatin nanoparticles are unsuitable as a vehicle but chitosan seemed to be a promising alternative. In this report we describe the production of stable 600-700-nm diameter nanoparticles of chitosan without organic solvents. Adsorption and release of bovine serum albumin seemed to be affected by the charge of the two reactants and at high doses not all adsorbate was released. PS4A, because of structural and compositional differences, had to be loaded on to the chitosan by freeze drying a suspension of the nanoparticles in a solution of the drug. After a rapid (burst) release phase, the rate of release into water was steady for the next 4 h, but not all the drug was released. In-vivo it was evident that PS4A and PS4alpha were equally active in solution or when formulated in the chitosan nanoparticles. These results show that chitosan nanoparticles, readily prepared without the use of organic solvents, are a suitable vehicle for the delivery of these immunostimulants from M. vaccae; the formulations might find application as antitumour agents.

Animals↗

PubMed1999

Gelatin behaviour in dilute aqueous solution: designing a nanoparticulate formulation.

Although it has been claimed that nanoparticles can be produced from gelatin, a naturally occurring polypeptide, the commercial conversion of animal collagen to gelatin results in a heterogeneous product with a wide molecular-weight range. This is probably responsible for the widely observed variation in the experimental conditions required for nanoparticle formation. In this study, 0.2% w/v aqueous B225 gelatin solutions were incubated under various conditions of time, temperature, pH and ethanol concentration and characterized by both size-exclusion high-performance liquid chromatography (HPLC) and dynamic light scattering. Gelatin was shown to be denatured when the temperature was increased to 37 degrees C (approx.) and the rate of renaturation was optimized over the temperature range 7-20 degrees C at pH 5.0, equivalent to the isoelectric point (IEP). The molecular-weight profile remained unchanged at 37 degrees C (approx.) in the pH range 5-7. When the gelatin solutions were mixed with ethanol, higher-molecular-weight fractions (microgel, delta and zeta fractions, all with molecular weights > 700 kDa) precipitated at ethanol concentrations lower than those required to precipitate the lower molecular weight material ( < 700 kDa), with maximum precipitation occurring close to the isoelectric point (pH 5.0). The molecular weight profile of gelatin in solution is evidently critically affected in a time-dependent manner by both pH and temperature. These two factors influence the noncovalent interactions responsible for the molecular structure of gelatin. The molecular weight profiles, in turn, affect the phase behaviour of gelatin in hydroalcoholic solutions. Systematically investigating the effect of time, temperature, pH and ethanol concentration on the molecular-weight-distribution profile of a gelatin solution enabled a robust method to be developed for the preparation of colloidal dispersions of non-aggregated gelatin nanoparticles 220-250 nm in diameter. This contrasts with the multiparticulate aggregates produced by earlier literature methods.

Chemistry, Pharmaceutical↗

PubMed1998

Fibronectin-binding peptides. I. Isolation and characterization of two unique fibronectin-binding peptides from gelatin.

Gelatin binds to fibronectin with a high affinity although the fibronectin-binding components have not been located. Fibronectin plays an important role in tumor cell metastasis and gelatin may have a profound effect on the metastatic process. In this study, fractionated acid-washed gelatin was cleaved with trypsin and resultant peptides fractionated by fibronectin-Sepharose affinity chromatography. After further purification using size exclusion HPLC and then reverse-phase HPLC, two unique peptides were obtained and sequenced. The binding affinities of these two peptides to fibronectin were evaluated by an ELISA method developed during this study and compared with the gelatin. Both possessed significantly higher binding affinities to fibronectin than gelatin alone.

Amino Acid Sequence↗

PubMed1998

Comparative measurement of the molecular weight of an antineoplastic glucan from BCG vaccine.

Bacillus Calmette-Guérin (BCG) vaccine, developed originally for the prophylaxis of tuberculosis, is a potent immunostimulant used to treat superficial bladder carcinoma in man. The aim of this study was to compare the molecular weight and self-association properties of an antineoplastic glucan (PS1A1) extracted from BCG vaccine as determined by different techniques including diffusion, light-scattering and chromatographic methods. In the diffusion experiments, a semi-empirical relationship was derived between the effective diffusion coefficients, Dp, and the weight-average molecular weights, Mw, of several dextrans used as standards, according to the equation Dp = 2.233 x 10(-6) x Mw(-0.66). On the basis of this relationship, the molecular weight of PS1A1 was found to be 57.4 kDa, although, unexpectedly, membrane association was high, most probably because of molecular branching. In the light-scattering experiment it was observed that, unlike dextran, PS1A1 undergoes concentration-dependent multimerization in water. However, the molecular weight of PS1A1 in 0.1 M sodium chloride ranged from 60 to 68 kDa, with a mean of 65 kDa, over the same concentration range. This value was in agreement with the molecular weight determined for PS1A1 by gel-filtration chromatography in previous studies, suggesting that 65 kDa represents the approximate monomeric size of the unassociated molecule. Thus, it was evident that the aggregation was suppressed by electrolyte. Elemental analysis by X-ray fluorescence showed that PS1A1 contained carbon, oxygen, hydrogen and phosphorus, indicating that hitherto unobserved ionized phosphate groups might promote electrostatic interactions.

Antineoplastic Agents↗

PubMed1998

Metastatic breast cancer presenting as heel pain.

The authors present a case of breast cancer metastasizing to the calcaneus that was confirmed by bone biopsy. The patient's complaint of heel pain provided the initial evidence of skeletal metastasis. Metastatic spread of cancer to the hand or foot (acrometastasis) is considered rare. However, the possibility of acrometastasis should be considered in any patient with a history of cancer presenting with skeletal pain, especially if the symptoms do not respond to therapy.

Adenocarcinoma↗

PubMed1997

Axotomy-induced apoptosis in adult rat primary sensory neurons.

Neuronal death following unilateral axotomy of a sensory nerve has long been inferred from neuronal counts of dorsal root ganglion neurons, using the contralateral ganglia as a control. The counting methods used usually involved the counting of neuronal nucleoli and made assumptions about them which could conceivably be flawed. Very few studies have used direct observations of dying or degenerating neurons to address questions concerning the duration of the period of neuronal death or the mechanisms involved in this process. Here we describe a morphological, morphometric and histochemical study into the nature and duration of sensory neuron death following transection and ligation of the sciatic nerve at mid-thigh level in the adult rat. We show that at least some of this neuronal loss occurs by apoptosis as defined by morphological criteria and in situ end-labelling of damaged DNA. Absolute numbers of apoptotic neurons were counted from serial paraffin sections of ganglia and estimates of neuronal numbers obtained by disector analysis at 1, 2, 3 and 6 months after axotomy. Using this approach we show that axotomy-induced apoptosis begins at around 1 week and continues up to at least 6 months after axotomy.

Animals↗

PubMed1997

Antineoplastic activity of BCG: location of antineoplastic glycans in the cellular integument of Mycobacterium bovis, BCG vaccine, Connaught substrain.

The polysaccharidic integument surrounding growing cells of attenuated Mycobacterium bovis Bacillus Calmette-Guérin (BCG) vaccine, Connaught substrain, can be removed with non-specific proteases. After 5 weeks incubation at 37 degrees C in Middlebrook 7H-9 medium, the collected cells were incubated with pronase and the integument and cells separated by centrifugation in a Ficoll-Histopaque preparation. After washing and drying, the detached integument accounted for 65% w/w of the original dried cell mass. Like the original cellular material, the detached integument manifested antineoplastic activity against a murine sarcoma model in vivo. Solubilization of the otherwise insoluble integument by boiling in water or by digestion in 8 M urea significantly enhanced activity of the integument itself, by 125 and 1,100 times, respectively. Integument extracts were shown to contain mainly glucose, with smaller quantities of other sugars, consistent with the presence in BCG of high molecular weight glycans, as previously reported. It is suggested that most, if not all, of the antineoplastic activity of BCG can be accounted for by the activity associated with the high-molecular weight polysaccharidic glycans which constitute the cellular integument.

Animals↗

PubMed1996

Sciatic nerve injury in the adult rat: comparison of effects on oligosaccharide, CGRP and GAP43 immunoreactivity in primary afferents following two types of trauma.

Using immunocytochemical and morphometric techniques, the localisation of three neuronal oligosaccharide antigens (two lactoseries and one globoseries oligosaccharide) were studied in the spinal cord and dorsal root ganglia of adult rats following unilateral crushing or transection of the sciatic nerve. The expression of CGRP and GAP43 was also studied for comparison. We found that following transection of the nerve the expression of lactoseries oligosaccharides and CGRP was permanently depressed, whilst that of the globoseries antigen (SSEA4) was unaffected. However following crush trauma and subsequent regeneration after 2 months, only the expression of one lactoseries antigen, LA4 remained significantly depressed. Our results suggest that different subsets of sensory neurons vary in the rate of reaction to injury and that one subset of neurons expressing a lactoseries oligosaccharide antigen is particularly susceptible to axotomy-induced changes. Furthermore neurons expressing the globoseries oligosaccharide antigen SSEA4 appear to be relatively unaffected by peripheral axotomy.

Afferent Pathways↗

PubMed1996

Chemical and ultrastructural investigations of Mycobacterium bovis BCG: implications for the molecular structure of the mycobacterial cell envelope.

The mycobacterial cell wall visualized by transmission electron microscopy (TEM) of thin sections of resin-embedded specimens is generally believed to consist of an electron-dense peptidoglycan, an electron-transparent arabinogalactanmycolate layer and an electron-dense outer layer (OL). In addition, a pseudocapsule known as the 'electron-transparent zone' (ETZ) has been observed after phagocytosis of mycobacteria by macrophages. TEM of thin sections of Mycobacterium bovis BCG, Tice substrain, revealed an OL bilayer, each of which measured 2-4 nm in diameter. The intermediate electron-transparent layer varied from 1 to about 250 nm in diameter and appears to be a previously observed oxygen-dependent amorphous integument that consists of hot water-extractable neutral polysaccharides, especially a recently characterized alpha glucan, comprising about 12% of the dry cell weight. This and other recent studies of BCG have revealed cell-surface features that may provide a better understanding of the outer mycobacterial cell envelope.

Cell Membrane↗

PubMed1996

Insoluble collagen matrices for prolonged delivery of proteins.

The purpose of this investigation was to evaluate the application of high molecular weight, insoluble collagen as a carrier material for proteins. Matrices were formulated and their behavior in buffer solution was investigated with focus on swelling and inner structure. Cross-linking with glutaraldehyde was introduced prior to the formation of the devices and its influence characterized. In addition, the enzymatic degradation process was studied and release experiments with systems loaded with fluorescent-labeled bovine serum albumin were carried out. Insoluble collagen matrices were characterized by intensive swelling in buffer resulting in development of a coarse porous character. Cross-linking strongly reduced the water penetration, leading to denser structures of the swollen devices. The continuous enzymatic degradation of the disk-shaped matrices by collagenase followed the kinetics of an heterogeneous enzymatic process with hindrance of proteolysis by the addition of glutaraldehyde. Release studies demonstrated that large amounts of model protein were held in the matrices with increased cross-linking degree. In presence of collagenase a prolonged release of the trapped protein over several days by matrix cleavage could be achieved. Insoluble collagen can be effective as a carrier material for proteins with an in vitro release characteristic by both diffusion-controlled and enzymatic degradation mechanisms. Cross-linking at the stage of preparing the aqueous dispersion offers an alternative to subsequent cross-linking processes.

Animals↗

PubMed1995

An anti-neoplastic glycan isolated from Mycobacterium bovis (BCG vaccine).

Tice substrain BCG is used clinically as an immunotherapeutic agent against superficial bladder cancer. A boiling-water extract of this BCG showed anti-tumour activity against a murine S180 sarcoma model and was fractionated into three fractions, A, B and C, by the use of Sephadex LH-20 chromatography. An anti-tumour glucan, PS1A1, was isolated from fraction PS1A with Sephadex G-75. The molecular mass of PS1A1 was between 65 and 87 kDa by Sephadex G-100 chromatography. The structure of PS1A1 was investigated by one- and two-dimensional NMR spectroscopy and methylation analysis and was demonstrated to be primarily 1-->6-alpha-linked glucose units. We postulate that the repeating unit is: [Formula: see text]

Animals↗

PubMed1995

The use of gelatin microparticles to delay the release of readily water-soluble materials.

The adsorption of D-arabinose onto gelatin microparticles demonstrated a Langmuirian adsorption pattern. Evaluation of the dissolution behaviour of D-arabinose-loaded gelatin microparticles suggested that the saccharide, loaded at a level below the adsorption saturation level, was released uniformly over a 14-h period after the loaded gelatin microparticles had been lyophilized for a second time. When dissolution curves were corrected for the initial burst effect seen after the gelatin microparticles had been loaded at higher levels of D-arabinose and lyophilized, steady-state release rates were also evident over prolonged periods. In addition, it was evident that the D-arabinose was adsorbed onto internal surfaces of the hydrated gelatin matrix. Calculation of this internal surface demonstrated the influence of the concentration of the glutaraldehyde used as a cross-linking agent and this parameter, in turn, influenced both the adsorption maxima and the subsequent equilibrium release rates. Application of this data base to a highly water-soluble complex polysaccharide antineoplastic agent, which has a higher molecular weight (22.4 kDa vs 150 Da), demonstrated similar behaviour in that a near zero-order release pattern over at least 16 h could be obtained by attention to the conditions under which the gelatin microparticles were made and subsequently loaded before lyophilization.

Adsorption↗