A study of the relationship between process conditions and mechanical strength of mineralized red algae in the preparation of a marine-derived bone void filler

Bone void fillers that can enhance biological function to augment skeletal repair have significant therapeutic potential in bone replacement surgery. This work focuses on the development of a unique microporous (0.5-10 mu m) marine-derived calcium phosphate bioceramic granule. It was prepared fro Corallina officinalis, a mineralized red alga, using a novel manufacturing process. This involved thermal processing, followed by a low pressure-temperature chemical synthesis reaction. The study found that the ability to maintain the unique algal morphology was dependent on the thermal processing conditions. This study investigates the effect of thermal heat treatment on the physiochemical properties of the alga. Thermogravimetric analysis was used to monitor its thermal decomposition. The resultant thermograms indicated the presence of a residual organic phase at temperatures below 500 degrees C and an irreversible solid-state phase transition from mg-rich-calcite to calcium oxide at temperatures over 850 degrees C. Algae and synthetic calcite were evaluated following heat treatment in an air-circulating furance at temperatures ranging from 400 to 800 degrees C. The highest levels of mass loss occurred between 400-500 degrees C and 700-800 degrees C, which were attributed to the organic and carbonate decomposition respectively. The changes in mechanical strength were quantified using a simple mechanical test, which measured the bulk compressive strength of the algae. The mechanical test used may provide a useful evaluation of the compressive properties of similar bone void fillers that are in granular form. The study concluded that soak temperatures in the range of 600 to 700 degrees C provided the optimum physiochemical properties as a precursor to conversion to hydroxyapatite (HA). At these temperatures, a partial phase transition to calcium oxide occurred and the original skeletal morphology of the alga remained intact.

RS-0406 arrests amyloid-B oligomer-induced behavioural deterioration in vivo

Clinically accessible compounds that arrest or reverse the effects of amyloid-ß (Aß) on progressively developing behavioural symptomatology and neuropathology in Alzheimer's disease (AD) have yet to become available. However, a viable strategy may be to target and neutralise soluble Aß oligomers, which have been shown to mediate synaptic dysfunction and to produce cognitive deficits in the intact organism. Inhibiting the aggregation of Aß is therapeutically attractive, as Aß aggregation is a pathological event and pharmacological interventions targeting this are likely to have a non-toxic profile. A behavioural assay, the alternating-lever cyclic-ratio schedule, was used to assess the effect of Aß oligomers and the non-peptide small molecule RS-0406 in male Sprague-Dawley rats. RS-0406 has been shown to inhibit Aß1-42 fibrillogenesis and protect against Aß1-42–induced cytotoxicity in primary hippocampal neurons. In the current study, RS-0406 ameliorated the adverse effects of secreted oligomers of human Aß on behaviour and dose dependently reduced the behavioural effects of Aß oligomers, with the highest dose, 10 µM, maintaining behaviour approximately at control levels. This effect appeared to be central; peripheral confounds having been extensively investigated. This is the first published report on the effects of RS-0406 in vivo and indicates that RS-0406 has potential as a pharmacotherapeutic intervention for behavioural deficits seen in the early stages of AD, and possibly as an intervention in the development of AD neuropathology. Indeed, an analogue of RS-0406 that could be administered peripherally might be a realistic candidate for the clinical treatment of AD.

Degradation and glycemic effects of His(7)-glucitol glucagon-like peptide-1(7-36)amide in obese diabetic ob/ob mice

Glucagon-like peptide-1(7-36)amide (tGLP-1) has attracted considerable potential as a possible therapeutic agent for type 2 diabetes. However, tGLP-1 is rapidly inactivated in vivo by the exopeptidase dipeptidyl peptidase IV (DPP IV), thereby terminating its insulin releasing activity. The present study has examined the ability of a novel analogue, His(7)-glucitol tGLP-1 to resist plasma degradation and enhance the insulin-releasing and antihyperglycemic activity of the peptide in 20-25-week-old obese diabetic ob/ob mice. Degradation of native tGLP-1 by incubation at 37 degreesC with obese mouse plasma was clearly evident after 3 h (35% intact). After 6 h, more than 87% of tGLP-1 was converted to GLP-1(9-36)amide and two further N-terminal fragments, GLP-1(7-28) and GLP-1(9-28). In contrast, His7-glucitol tGLP-1 was completely resistant to N-terminal degradation. The formation of GLP-1(9-36)amide from native tGLP-1 was almost totally abolished by addition of diprotin A, a specific inhibitor of DPP IV. Effects of tGLP-1 and His7-glucitol tGLP-1 were examined in overnight fasted obese mice following i.p. injection of either peptide (30 nmol/kg) together with glucose (18 mmol/kg) or in association with feeding. Plasma glucose was significantly lower and insulin response greater following administration of His7-glucitol tGLP-1 as compared to glucose alone. Native tGLP-1 lacked antidiabetic effects under the conditions employed, and neither peptide influenced the glucose-lowering action of exogenous insulin (50 units/kg). Twice daily s.c. injection of ob/ob mice with His(7)-glucitol tGLP-1 (10 nmol/kg) for 7 days reduced fasting hyperglycemia and greatly augmented the plasma insulin response to the peptides given in association with feeding. These data demonstrate that His(7)-glucitol tGLP-1 displays resistance to plasma DPP IV degradation and exhibits antihyperglycemic activity and substantially enhanced insulin-releasing action in a commonly used animal model of type 2 diabetes. (C) 2001 Elsevier Science B.V. All rights reserved.

Improved glycaemic control in obese diabetic ob/ob mice using N-terminally modified gastric inhibitory polypeptide

Gastric inhibitory polypeptide (GIP) is an important insulin-releasing hormone of the enteroinsular axis which is rapidly inactivated by the exopeptidase dipeptidyl peptidase (DPP) IV. The present study has examined the ability of Tyr(1)-glucitol GIP to be protected from plasma degradation and to enhance insulin-releasing and antihyperglycaemic activity in 20- to 25-week-old obese diabetic ob/ob mice. Degradation of GIP by incubation at 37 degrees C with obese mouse plasma was clearly evident after 3 h (35% degraded). After 6 h, more than 61% of GIP was converted to GIP(3-42) whereas N-terminally modified Tyr(1)-glucitol GIP was resistant to degradation in plasma (>99% intact after 6 h). The formation of GIP(3-42) was almost completely abolished by inhibition of plasma DPP IV with diprotin A. Effects of GIP and Tyr(1)-glucitol GIP were examined in overnight-fasted obese mice following i.p. injection of either peptide (20 nmol/kg) together with glucose (18 mmol/kg) or in association with feeding. Most prominent effects were observed in the former group where plasma glucose values at 60 min together with the area under the curve (AUC) for glucose were significantly lower following GIP (AUC, 874 +/- 72 mmol/l.min; P

N-terminally modified glucagon-like peptide-1(7-36) amide exhibits resistance to enzymatic degradation while maintaining its antihyperglycaemic activity in vivo

Glucagon-like peptide-1 (7-36)amide (tGLP-1) is inactivated by dipeptidyl peptidase (DPP) IV by removal of the NH2-terminal dipeptide His(7)-Ala(8). We examined the degradation of NH2-terminally modified His(7)-glucitol tGLP-1 and its insulin-releasing and antihyperglycaemic activity in vivo, tGLP-1 was degraded by purified DPP IV after 4 h (43% intact) and after 12 hi 89% was converted to GLP-1(9-36)amide. In contrast > 99% of His(7)-glucitol tGLP-1 remained intact at 12 h. His(7)-glucitol tGLP-1 was similarly resistant to plasma degradation in vitro. His7-glucitol tGLP-1 showed greater resistance to degradation in vivo (92% intact) compared to tGLP-1 (27% intact) 10 min after i.p. administration to Wistar rats. Glucose homeostasis was examined following i.p. injection of both peptides (12 nmol/kg) together with glucose (18 mmol/kg). Plasma glucose concentrations were significantly reduced and insulin concentrations elevated following peptides administration compared with glucose alone. The area under the curve (AUC) for glucose for controls (AUC 691 +/- 35 mM/min) was significantly lower after administration of tGLP-1 and His7-glucitol tGLP-1 (36 and 49% less; AUC; 440 +/- 40 and 353 +/- 31 mM/min, respectively; P

Jettisoning Ballast or Fuel? Caudal Autotomy and Locomotory Energetics of the Cape Dwarf Gecko Lygodactylus capensis (Gekkonidae)

Many lizard species will shed their tail as a defensive response (e.g., to escape a putative predator or aggressive conspecific). This caudal autotomy incurs a number of costs as a result of loss of the tail itself, loss of resources (i.e., stored in the tail or due to the cost of regeneration), and altered behavior. Few studies have examined the metabolic costs of caudal autotomy. A previous study demonstrated that geckos can move faster after tail loss as a result of reduced weight or friction with the substrate; however, there are no data for the effects of caudal autotomy on locomotory energetics. We examined the effect of tail loss on locomotory costs in the Cape dwarf gecko Lygodactylus capensis (similar to 0.9 g) using a novel method for collecting data on small lizards, a method previously used for arthropods. We measured CO2 production during 5-10 min of exhaustive exercise (in response to stimulus) and during a 45-min recovery period. During exercise, we measured speed (for each meter moved) as well as total distance traveled. Contrary to our expectations, tailless geckos overall expended less effort in escape running, moving both slower and for a shorter distance, compared with when they were intact. Tailless geckos also exhibited lower excess CO2 production (CO2 production in excess of normal resting metabolic rate) during exercising. This may be due to reduced metabolically active tissue (tails represent 8.7% of their initial body mass). An alternative suggestion is that a change in energy substrate use may take place after tail loss. This is an intriguing finding that warrants future biochemical investigation before we can predict the relative costs of tail loss that lizards might experience under natural conditions.

Histological and ultrastructural investigation of retinal microaneurysm development in diabetic patients

BACKGROUND: Although microaneurysms are a clinicopathological hallmark of diabetic retinopathy, there have been few ultrastructural studies of these important lesions. As a result, knowledge of the mechanisms involved in the pathogenesis of microaneurysms remains fragmentary. This study provides histological and ultrastructural evidence of various stages in microaneurysm formation within the retinal vasculature. METHODS: The eyes of three type II diabetic patients, obtained within 24 hours of death, were studied by the trypsin digest technique. Eyes from two further type II diabetics were fixed in 2.5% glutaraldehyde within 12 hours of death and processed for electron microscopy. RESULTS: In the trypsin digest preparations, small saccular and fusiform microaneurysms were observed in the peripheral retinal. In the central retina, the microaneurysms ranged in morphology from thin walled, cellular forms to dense, acellular, hyalinised forms. Ultrastructurally, four distinct groups of microaneurysm were observed. Type I showed an extensive accumulation of polymorphonuclear cells into the lumen. The endothelium remained intact, although pericytes were invariably absent. Type II microaneurysms were typified by large numbers of red blood cells (RBCs) in the lumen. Endothelial cells and pericytes were completely absent. The type III microaneurysm was also non-perfused and contained aggregates of irregularly shaped RBC profiles and RBC breakdown products. Recanalisation by new vessels into the occluded lumen was observed in one microaneurysm. Type IV microaneurysms were almost or completely sclerosed, with extensive fibrosis and lipid infiltration into the lumen and basement membrane wall. CONCLUSION: This investigation describes several distinctive stages in the formation of microaneurysms during diabetic retinopathy. With reference to the pathogenesis of retinal microaneurysms, the interaction of various cell types is discussed and the significance of vascular cell death and localised hypertensive events highlighted.

Increased endocytosis in retinal vascular endothelial cells grown in high glucose medium is modulated by inhibitors of nonenzymatic glycosylation

We sought to determine if hyperglycaemia is responsible for increased retinal vascular endothelial-cell (RVEC) endocytosis in diabetes and to assess the role of nonenzymatic glycosylation in mediation of this novel endothelial-cell pathology. RVECs were propagated in media containing either 5 or 25 mmol/l glucose for up to 10 days after which they were exposed to the protein tracer horseradish peroxidase for 30 min. The level of RVEC endocytosis was quantified in intact cell monolayers by electron microscopic stereology, and in cell lysates by a simple spectrophotometric method. The effect of the nonenzymatic glycosylation inhibitors, aminoguanidine and D-lysine, on high-glucose medium induced changes in RVEC endocytosis was tested by inclusion of these agents in the culture medium. RVECs exposed to 25 mmol/l glucose showed a stepwise increase in endocytosis of horseradish peroxidase culminating in a two- to threefold increase after 10 days. Endocytosis returned to normal levels after a further 10 days in 5 mmol/l glucose medium. The increase in RVEC endocytosis was markedly reduced, but not completely normalised, by aminoguanidine and D-lysine. Exposure of cultured RVECs to 25 mmol/l glucose causes an increase in endocytosis of similar magnitude to that experienced by RVEC in early diabetes, and implicates hyperglycaemia in the latter situation. A significant component of the increase in RVEC endocytosis appears to be mediated by nonenzymatic glycosylation.

Fasciola hepatica: Disruption of the vitelline cells in vitro by the sulphoxide metabolite of triclabendazole

The effects of the active sulphoxide metabolite of the fasciolicide triclabendazole (Fasinex, Ciba-Geigy) on the vitelline cells of Fusciola hepatica were determined in vitro by transmission electron microscopy using both intact flukes and tissue-slice material. At a triclabendazole concentration of 15 mu g/ml the vitelline cells of intact flukes showed ultrastructural changes only after prolonged incubation periods (12-24 h). The changes observed were a swelling of the granular endoplasmic reticulum (GER) cisternae with decreased ribosomal covering in the intermediate-type cells and condensation of chromatin and disappearance of the nucleolus in the nucleus of the stem cell. Similar changes were evident more quickly (by 6 h) in whole flukes treated at the higher concentration of 50 mu g/ml. The shell globule clusters were loosely packed in the intermediate type-2 cells, and the number of intermediate type-1 cells declined with more prolonged incubation. Disruption of the nurse-cell cytoplasm was also observed from 12 h onwards. After only 6 h incubation of tissue-slice material at 50 mu g/ml, intermediate type-1 cells were absent, shell globule clusters in mature cells were loosely packed and the nurses cell cytoplasm was badly disrupted. By 12 h the vitelline cells were vacuolated and grossly abnormal. The results are discussed in relation to postulated actions of triclabendazole against the microtubule component of the cytoskeleton and against protein synthesis in the fluke.

Are there clinical features of a sensitized cough reflex?

Cough reflex hypersensitization is a key feature in patients with troublesome cough. The clinical consequence of this hypersensitive state is typified by bouts of coughing often triggered by low threshold stimuli encountered by the patient during normal daily activities including exposure to aerosols, scents and odours, a change in air temperature and when talking or laughing. These features are often perceived by cough patients to be the most disruptive aspect of their condition and undoubtedly contribute to impaired quality of life. Patients with troublesome cough may describe a range of additional symptoms and sensations including an 'urge to cough' or the feeling of an 'itch' at the back of the throat, or a choking sensation and occasionally chest pain or breathlessness. It is uncertain if these features arise due to the processes responsible for cough reflex sensitization or as a direct consequence of the underlying cough aetiology. In an attempt to understand the clinical features of a sensitized cough reflex, the spectrum of symptoms typically described by cough patients will be reviewed and possible underlying mechanisms considered. Since an intact cough reflex is crucial to airway protection, anti-tussive treatment that attenuates the hypersensitive cough state rather than abolishing the cough reflex completely would be preferable. Identifying such agents remains a clinical, scientific and pharmacological challenge. (c) 2008 Elsevier Ltd. All rights reserved.

Interaction of prothrombin with a phospholipid surface: evidence for a membrane-induced conformational change

Prothrombin interacts with phosphatidylserine containing platelet membranes via its N-terminal, gamma-carboxyglutamate (gla) residue-rich domain. Once bound it is cleaved to form the active protease, thrombin (factor IIa). Human prothrombin was cleaved with cathepsin G in the absence of calcium and magnesium ions. Under these conditions, the gla domain was removed. Phospholipid protected the protein from this proteolytic event, and this suggests that a conformational change may be induced by interaction with phospholipids. Binding of prothrombin to a surface containing 20% phosphatidylserine/80% phosphatidylcholine was detected by surface plasmon resonance, whereas no interaction with gla-domainless prothrombin was observed. Binding of intact prothrombin in the presence of calcium ions showed complex association kinetics, suggesting multiple modes of initial interaction with the surface. The kinetics of the dissociation phase could be fitted to a two-phase, exponential decay. This implies that there are at least two forms of the protein on the surface one of which dissociates tenfold more slowly than the other. Taken together, these data suggest that, on binding to a membrane surface, prothrombin undergoes a conformational change to a form which binds more tightly to the membrane.

Body mass-abundance relationships are robust to cascading effects in marine food webs

Body mass has been shown to scale negatively with abundance in a wide range of habitats and ecosystems. It is believed that this relationship has important consequences for the distribution and maintenance of energy in natural communities. Some studies have shown that the relationship between body mass and abundance may be robust to major food web perturbations, fuelling the belief that natural processes may preserve the slope of this relationship and the associated cycling of energy and nutrients. Here, we use data from a long-term experimental food web manipulation to examine this issue in a semi-natural environment. Similar communities were developed in large experimental mesocosms over a six month period. Some of the mesocosms were then subjected to species removals, based on the mean strength of their trophic interactions in the communities. In treatments where the strongest interactors were removed, a community-level trophic cascade occurred. The biomass density of invertebrates increased dramatically in these communities, which led to a suppression of primary production. In spite of these widespread changes in ecosystem functioning, the slope of the relationship between body mass and abundance remained unchanged. This was the case whether average species body mass and abundance or individual organism size spectra were considered. An examination of changes in species composition before and after the experimental manipulations revealed an important mechanism for maintaining the body mass-abundance relationship. The manipulated communities all had a higher species turnover than the intact communities, with the highest turnover in communities that experienced cascading effects. As some species increased in body mass and abundance, new species filled the available size-abundance niches that were created. This maintained the overall body mass-abundance relationship and provided a stabilising structure to these experimental communities.

Enantioselective dioxygenase-catalysed formation and thermal racemisation of chiral thiophene sulfoxides

Substituted chiral thiophene 1-oxides and their cycloadducts of variable enantiopurity have been isolated as products of dioxygenase-catalysed sulfoxidation of the corresponding thiophenes using intact cells of Pseudomonas putida; thermal racemization (Delta G(double dagger) = 25.1 kcal mol(-1)) of the enantiopure metabolite (1R)-2-methylbenzo[b]thiophene 1-oxide has been observed.

THE EFFECT OF THE SULFOXIDE METABOLITE OF TRICLABENDAZOLE (FASINEX) ON THE TEGUMENT OF MATURE AND IMMATURE STAGES OF THE LIVER FLUKE, FASCIOLA-HEPATICA

The effects of the novel benzimidazole, triclabendazole (TCBZ) ('Fasinex', Ciba-Geigy), in its active sulphoxide metabolite form (TCBZ-SX), on the tegumental ultrastructure of Fasciola hepatica were determined in vitro by transmission electron microscopy (TEM), using both intact flukes and tissue-slice material. At a concentration of 15 mu g/ml, the tegument of the whole adult fluke showed ultrastructural changes only after prolonged time-periods, with vacuolation at the base of the syncytium and accumulation of T2 secretory bodies in the tegumental cells. At a concentration of 50 mu g/ml, with both whole flukes and tissue-slices, the tegument appeared extremely abnormal with accumulation of secretory bodies towards the base of the syncytium. With longer incubation times, the tegument was completely sloughed away and the tegumental cells became synthetically inactive. The tegument of the 3-week-old juvenile became progressively convoluted at the apex, while in the basal regions there was severe vacuolation. In the tegumental cells, there were accumulations of T1 secretory bodies. These results confirm TCBZ as a potent fasciolicide, being very effective in disrupting the fluke tegument. They may go some way to explain the mode of action of this important fasciolicide.

FASCIOLA-HEPATICA - THE EFFECT OF THE MICROTUBULE INHIBITORS COLCHICINE AND TUBULOZOLE-C ON THE ULTRASTRUCTURE OF THE ADULT FLUKE

The effect of the microtubule inhibitors colchicine (1 x 10(-3) M) and tubulozole-C(1 x 10(-6) M) on the ultrastructure of adult Fasciola hepatica has been determined in vitro by transmission electron microscopy (TEM), using both intact flukes and tissue-slice material. With colchicine treatment, the apical membrane of the tegument became increasingly convoluted and blebbed, while accumulations of T1 secretory bodies occurred in the basal region of the syncytium, leading to progressively fewer secretory bodies in the syncytium. In the tegumental cells there were distinct accumulations of Tl secretory bodies around the Golgi complexes, which remained active for up to 12 h incubation. Tubulozole-treated flukes showed more severe effects, with initial accumulations of secretory bodies, both at the tegumental apex and base. This was followed in the later time-periods by the sloughing of the tegumental syncytium. In the underlying tegumental cells, the granular endoplasmic reticulum (GER) cisternae were swollen and disrupted, becoming concentrated around the nucleus. The Golgi complexes were dispersed to the periphery of the cells and gradually disappeared from the cytoplasm. After treatment with both drugs, the cell population in the vitelline follicles was altered, with an abnormally large proportion of stem cells and relatively few intermediate type 1 cells. The nurse cell cytoplasm became fragmented and was no longer in contact with the vitelline cells, while the shell globule clusters within the intermediate type 2 and mature cells were loosely packed. In the mature vitelline cells, 'yolk' globules and glycogen deposits became fewer than normal and lipid droplets were observed. The results are discussed in relation to the different modes of action of the two drugs and potential significance of this to anthelmintic (benzimidazole) therapy.