Hydrogel oxygen reservoirs increase functional integration of neural stem cell grafts by meeting metabolic demands.

Injectable biomimetic hydrogels have great potential for use in regenerative medicine as cellular delivery vectors. However, they can suffer from issues relating to hypoxia, including poor cell survival, differentiation, and functional integration owing to the lack of an established vascular network. Here we engineer a hybrid myoglobin:peptide hydrogel that can concomitantly deliver stem cells and oxygen to the brain to support engraftment until vascularisation can occur naturally. We show that this hybrid hydrogel can modulate cell fate specification within progenitor cell grafts, resulting in a significant increase in neuronal differentiation. We find that the addition of myoglobin to the hydrogel results in more extensive innervation within the host tissue from the grafted cells, which is essential for neuronal replacement strategies to ensure functional synaptic connectivity. This approach could result in greater functional integration of stem cell-derived grafts for the treatment of neural injuries and diseases affecting the central and peripheral nervous systems.

Improvement of Storage Medium for Cultured Human Retinal Pigment Epithelial Cells Using Factorial Design.

Storage of human retinal pigment epithelium (hRPE) can contribute to the advancement of cell-based RPE replacement therapies. The present study aimed to improve the quality of stored hRPE cultures by identifying storage medium additives that, alone or in combination, contribute to enhancing cell viability while preserving morphology and phenotype. hRPE cells were cultured in the presence of the silk protein sericin until pigmentation. Cells were then stored for 10 days in storage medium plus sericin and either one of 46 different additives. Individual effects of each additive on cell viability were assessed using epifluorescence microscopy. Factorial design identified promising additive combinations by extrapolating their individual effects. Supplementing the storage medium with sericin combined with adenosine, L-ascorbic acid and allopurinol resulted in the highest cell viability (98.6 ± 0.5%) after storage for three days, as measured by epifluorescence microscopy. Flow cytometry validated the findings. Proteomics identified 61 upregulated and 65 downregulated proteins in this storage group compared to the unstored control. Transmission electron microscopy demonstrated the presence of melanosomes after storage in the optimized medium. We conclude that the combination of adenosine, L-ascorbic acid, allopurinol and sericin in minimal essential medium preserves RPE pigmentation while maintaining cell viability during storage.

Constrained evolution of a bispecific enzyme: lessons for biocatalyst design.

One of the central goals of protein design and engineering is to be able to accurately predict the effects of a mutation on stability and activity. However, the genetic context into which mutations are introduced can lead to complex interactions between the mutation and other amino acids and unpredictable, non-additive, effects. This phenomenon is known as intramolecular epistasis and has been shown to restrict evolutionary paths through laboratory directed evolution experiments and ancestral protein reconstruction, but has rarely been studied at a quantitative level in naturally evolving enzymes. Atrazine-specific and atrazine/ametryn bispecific triazine hydrolases (TrzN) have evolved in different bacterial strains over the past fifty years in response to the presence of the synthetic herbicides atrazine and ametryn. Here, we have investigated all 24 evolutionary trajectories that are possible from monofunctional to bispecific TrzN isoforms in terms of activity, stability, expression and structure. The results reveal that half of these trajectories are unviable due to inactive intermediates, with only 1/24 trajectories exhibiting consistent improvement in bispecificity. The most viable path requires the mutation of Gln241 to Glu241 first, which increases activity 3-fold with atrazine and 105-fold with ametryn, which is further optimised in subsequent evolutionary steps. The epistatic interactions between mutations, involving control of the pKa of catalytic residues, the thermostability of the protein, and soluble expression are shown to be responsible for the bottlenecks in this evolutionary landscape. This comprehensive analysis of the evolution of bispecificity highlights the importance of epistasis in protein engineering and evolution, which makes identifying the correct sequence in which to combine mutations extremely important.

Effect of activated protein C in second intention healing of equine distal limb wounds: a preliminary study.

OBJECTIVE: To investigate the effect of activated protein C (APC) on second intention healing of distal limb wounds in horses. METHODS: In this experimental study of eight Standardbred geldings, six full-thickness skin wounds (2 × 1.5 cm) were created on one metacarpus (biopsy limb) and five similar wounds were created on the contralateral metacarpus (photographed limb). Three wounds on the biopsy limb were treated topically with 190 µg APC on days 1, 3, 6 and 9, while the remaining three wounds were untreated (control). One treated and one control wound were biopsied on days 4, 7 and 11 for histopathology. Wounds on the photographed limb were treated with either 66% Manuka honey gel, a commercial antibiotic ointment (bacitracin-neomycin-polymixin B ointment; BNP) or petrolatum daily throughout healing, treated on days 1,3,6 and 9 with 190 µg APC or left untreated. These wounds were digitally photographed and the wound area measured on day 1, then weekly until day 49. Overall time to healing was recorded. RESULTS: There was no effect of APC on wound size, the rate of healing or the overall time to heal. However, compared with control wounds, histological scoring demonstrated enhanced epithelialisation (day 4) and angiogenesis (day 11). Wound healing variables for wounds treated with APC, Manuka honey gel and control wounds were not different and the variables for wounds treated with BNP and petrolatum demonstrated delayed healing. CONCLUSION: The improvements in histological scores in APC-treated wounds suggest further study into the effect of APC on second intention wound healing in horses is warranted.

Low circulating protein C levels are associated with lower leg ulcers in patients with diabetes.

Activated protein C (APC) promotes angiogenesis and reepithelialisation and accelerates healing of diabetic ulcers. The aim of this study was to determine the relationship between the incidence of lower leg ulcers and plasma levels of APC's precursor, protein C (PC), in diabetic patients. Patients with diabetes who had a lower leg ulcer(s) for >6 months (n = 36) were compared with age-, type of diabetes-, and sex-matched subjects with diabetes but without an ulcer (n = 36, controls). Total PC was assessed using a routine PC colorimetric assay. There was a significantly (P < 0.001) lower level of plasma PC in patients with ulcers (103.3 ± 22.7, mean ± SD) compared with control (127.1 ± 34.0) subjects, when corrected for age and matched for gender and type of diabetes. Ulcer type (neuropathic, ischaemic, or mixed) was not a significant covariate for plasma PC levels (P = 0.35). There was no correlation between PC levels and gender, type of diabetes, HbA1c, or C-reactive protein in either group. In summary, decreased circulating PC levels are associated with, and may predispose to, lower leg ulceration in patients with diabetes.

Enzymatic modification of regenerated cellulosic fabrics to improve bacteria sorption properties.

This research investigates the effect of enzymatic treatment of two different regenerated cellulosic fibers (Lyocell and viscose) on their ability of bacteria sorption from an aqueous suspension. The sorption of Escherichia coli (E. coli, Gram negative) and Staphylococcus aureus (S. aureus, Gram positive) cells by treated Lyocell and viscose fabrics were determined by measuring the optical density (OD) of the remaining bacteria suspension after removal of the fabric samples using spectrometry. Fourier transform infrared spectroscopy and scanning electron microscopy (SEM) were utilized to investigate structural and morphological changes of the enzyme treated samples. The result showed that the moisture content and crystallinity of both viscose and Lyocell samples increased after enzymatic treatment. Comparing the results of OD measurements indicated that enzymatic treatment of cellulosic samples significantly increased the bacteria absorption properties compared to those untreated samples. However, treated samples showed different ranges of sorption ability with different kinds of bacteria. The maximum bacteria sorption of 38% and 37% of E. coli bacteria from an aqueous suspension was found for the treated viscose and Lyocell samples compared with only 20% and 10% of the untreated viscose and Lyocell samples, respectively. It was also found that S. aureus sorption of cellulose-treated viscose and Lyocell fabrics from a bacterial suspension could significantly improve up to 33% compared with only 5% of untreated samples. Furthermore, SEM micrographs confirmed that bacterial sorption of the cellulose-treated samples were effectively improved in terms of their uniform sorption on the fibers surface.

SIRT1, a class III histone deacetylase, regulates TNF-α-induced inflammation in human chondrocytes.

OBJECTIVE: The present study was performed to elucidate the possible role of SIRT1 signaling in joint inflammation in human articular chondrocytes. DESIGN: Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western blotting were performed to detect gene products and proteins involved in tumor necrosis factor α (TNF-α)-induced inflammation and cartilage degradation in human primary chondrocytes. Matrix metalloproteinase (MMP)-2 and MMP-9 activity was evaluated by gelatin zymography. Overexpression and knockdown of SIRT1 were also performed to investigate whether SIRT1 is associated with the anti-inflammatory activity of resveratrol in chondrocytes. RESULTS: Resveratrol dose-dependently inhibited TNF-α-induced cyclooxygenase-2 (COX-2), MMP-1, MMP-3, MMP-13 and PGE(2) production in human chondrocytes. Moreover, MMP-2 and MMP-9 activity was increased by treatment with TNF-α; however, SIRT1 activation decreased the proinflammatory effects induced by TNF-α. In addition, treatment of SIRT1 activator and overexpression of SIRT1 inhibited the expression and activation of the main proinflammatory regulator NF-κB, which was increased by TNF-α. When SIRT1 was overexpressed in chondrocytes, the anti-inflammatory action of SIRT1 was similar to that exerted by resveratrol. CONCLUSIONS: SIRT1 activation deacetylates and inactivates NF-κB, and thereby, exerts an anti-inflammatory effect on chondrocytes, suggesting that SIRT1 activators could be explored as potential treatments for arthritis.

The mitochondrial genome and transcriptome of the basal dinoflagellate Hematodinium sp.: character evolution within the highly derived mitochondrial genomes of dinoflagellates.

The sister phyla dinoflagellates and apicomplexans inherited a drastically reduced mitochondrial genome (mitochondrial DNA, mtDNA) containing only three protein-coding (cob, cox1, and cox3) genes and two ribosomal RNA (rRNA) genes. In apicomplexans, single copies of these genes are encoded on the smallest known mtDNA chromosome (6 kb). In dinoflagellates, however, the genome has undergone further substantial modifications, including massive genome amplification and recombination resulting in multiple copies of each gene and gene fragments linked in numerous combinations. Furthermore, protein-encoding genes have lost standard stop codons, trans-splicing of messenger RNAs (mRNAs) is required to generate complete cox3 transcripts, and extensive RNA editing recodes most genes. From taxa investigated to date, it is unclear when many of these unusual dinoflagellate mtDNA characters evolved. To address this question, we investigated the mitochondrial genome and transcriptome character states of the deep branching dinoflagellate Hematodinium sp. Genomic data show that like later-branching dinoflagellates Hematodinium sp. also contains an inflated, heavily recombined genome of multicopy genes and gene fragments. Although stop codons are also lacking for cox1 and cob, cox3 still encodes a conventional stop codon. Extensive editing of mRNAs also occurs in Hematodinium sp. The mtDNA of basal dinoflagellate Hematodinium sp. indicates that much of the mtDNA modification in dinoflagellates occurred early in this lineage, including genome amplification and recombination, and decreased use of standard stop codons. Trans-splicing, on the other hand, occurred after Hematodinium sp. diverged. Only RNA editing presents a nonlinear pattern of evolution in dinoflagellates as this process occurs in Hematodinium sp. but is absent in some later-branching taxa indicating that this process was either lost in some lineages or developed more than once during the evolution of the highly unusual dinoflagellate mtDNA.

Expression of bacterial levanase in yeast enables simultaneous saccharification and fermentation of grass juice to bioethanol.

This study demonstrates use of recombinant yeast to simultaneously saccharify and ferment grass juice (GJ) to bioethanol. A modified Bacillus subtilis levanase gene (sacC) in which the native bacterial signal sequence was replaced with a yeast α-factor domain, was synthesised with yeast codon preferences and transformed into Saccharomyces cerevisiae (strain AH22) using the expression vector pMA91. AH22:psacC transformants secreted sacCp as an active, hyper-glycosylated (>180 kDa) protein allowing them to utilise inulin (ß[2-1] linked fructose) and levan (ß[2-6] linkages) as growth substrates. The control (AH22:pMA91) strain, transformed with empty plasmid DNA was not able to utilise inulin or levan. When cultured on untreated GJ levels of growth and bioethanol production were significantly higher in experiments with AH22:psacC than with AH22:pMA91. Bioethanol yields from AH22:psacC grown on GJ (32.7[±4] mg mL(-1)) compared closely to those recently achieved (Martel et al., 2010) using enzymatically pre-hydrolysed GJ (36.8[±4] mg mL(-1)).

Expression, purification and use of the soluble domain of Lactobacillus paracasei beta-fructosidase to optimise production of bioethanol from grass fructans.

Microbial inulinases find application in food, pharmaceutical and biofuel industries. Here, a novel Lactobacillus paracasei beta-fructosidase was overexpressed as truncated cytosolic protein ((t)fosEp) in Escherichia coli. Purified (t)fosEp was thermostable (10-50 degrees C) with a pH optimum of 5; it showed highest affinity for bacterial levan (beta[2-6] linked fructose) followed by nystose, chicory inulin, 1-kestose (beta[2-1] linkages) and sucrose (K(m) values of 0.5, 15, 15.6, 49 and 398 mM, respectively). Hydrolysis of polyfructose moieties in agriculturally-sourced grass juice (GJ) with (t)fosEp resulted in the release of >13 mg/ml more bioavailable fructose than was measured in untreated GJ. Bioethanol yields from fermentation experiments with Brewer's yeast and GJ+(t)fosEp were >25% higher than those achieved using untreated GJ feedstock (36.5[+/-4.3] and 28.2[+/-2.7]mg ethanol/ml, respectively). This constitutes the first specific study of the potential to ferment ethanol from grass juice and the utility of a novel core domain of beta-fructosidase from L. paracasei.

Conformational sampling, catalysis, and evolution of the bacterial phosphotriesterase.

To efficiently catalyze a chemical reaction, enzymes are required to maintain fast rates for formation of the Michaelis complex, the chemical reaction and product release. These distinct demands could be satisfied via fluctuation between different conformational substates (CSs) with unique configurations and catalytic properties. However, there is debate as to how these rapid conformational changes, or dynamics, exactly affect catalysis. As a model system, we have studied bacterial phosphotriesterase (PTE), which catalyzes the hydrolysis of the pesticide paraoxon at rates limited by a physical barrier-either substrate diffusion or conformational change. The mechanism of paraoxon hydrolysis is understood in detail and is based on a single, dominant, enzyme conformation. However, the other aspects of substrate turnover (substrate binding and product release), although possibly rate-limiting, have received relatively little attention. This work identifies "open" and "closed" CSs in PTE and dominant structural transition in the enzyme that links them. The closed state is optimally preorganized for paraoxon hydrolysis, but seems to block access to/from the active site. In contrast, the open CS enables access to the active site but is poorly organized for hydrolysis. Analysis of the structural and kinetic effects of mutations distant from the active site suggests that remote mutations affect the turnover rate by altering the conformational landscape.

Referral and attendance at a specialist antenatal clinic: qualitative study of women's views.

OBJECTIVE: To explore women's views on being referred to and attending a specialist antenatal hypertension clinic. DESIGN: Qualitative interview study. SETTING: A pregnancy hypertension clinic in a large teaching hospital in the East Midlands. POPULATION: Twenty-one women (aged 18 years and above) attending the pregnancy hypertension clinic for the first time during their current pregnancy. METHODS: Women who had been referred to and attended a specialist antenatal clinic participated in semi-structured interviews. Data analysis was based on the constant comparative method. MAIN OUTCOME MEASURES: Women's experiences and perceptions of being referred to and attending a specialist antenatal clinic. RESULTS: Being referred to the clinic conferred an 'at risk' status on women. Some women welcomed the referral but others experienced it as unsettling. Many were unclear about why they had been identified as being at risk or had difficulties in accepting the legitimacy of the reason for referral. Women were often inadequately informed about why they were referred to the clinic, what they could expect and the benefits of attending the clinic over management in the community. Although attendance at the clinic was cited as a source of reassurance, the reassurance was often made necessary by concern raised by the initial referral. CONCLUSIONS: Women's accounts suggest that the interface between community and secondary antenatal services needs improvement to minimise possible adverse effects from identifying women as being 'at risk' during pregnancy.

Participating in a trial in a critical situation: a qualitative study in pregnancy.

BACKGROUND: Randomised controlled trials of interventions in critical situations are necessary to establish safety and evaluate outcomes. Pregnant women have been identified as a potentially vulnerable population. OBJECTIVE: To explore women's experiences of being recruited to ORACLE, a randomised controlled trial of antibiotics in pre-term labour. METHODS: Twenty qualitative interviews were conducted with women who had participated in ORACLE. Analysis was based on the constant comparative method. RESULTS: Women gave prominence to the socioemotional aspects of their interactions with healthcare professionals in making decisions on trial participation. Comments on the quality of written and spoken information were generally favourable, but women's accounts suggest that the stressful nature of the situation affected their ability to absorb the information. Women generally had poor understanding of trial design and practices. The main motivation for trial participation was the possibility of an improved outcome for the baby. The second and less prominent motivation was the opportunity to help others, but this was conditional on there being no risks associated with trial participation. In judging the risks of participation, women seemed to draw on "common sense" understandings including a perception that antibiotics were risk free. DISCUSSION: Recruitment to trials in critical situations raises important questions. Future studies should explore how rigorous governance arrangements for trials, particularly in critical situations, can protect participants rather than relying on ideals of informed consent that may be impossible to achieve. Future research should include a focus on interactions between research candidates and professionals involved in recruitment.

African trypanosomiasis in a British soldier.

BACKGROUND: Human African trypanosomiasis (sleeping sickness) is a parasitic infection transmitted by day-biting tsetse flies. The diagnostic gold standard is microscopy of blood, lymph node aspirates or CSF. The disease is invariably fatal, if not treated. There are over 300 000 new cases of sleeping sickness each year, and approximately 100,000 deaths. CASE PRESENTATION: We describe a British soldier who acquired trypanosomiasis in Malawi. He gave no history of a painful insect bite but presented with classical early signs of sleeping sickness (a primary chancre, regional lymphadenopathy, circinate erythema and a cyclical fever pattern). His condition worsened in the next week and trypanosomes were observed in a blood sample. He was aeromedically evacuated to Johannesburg, where Stage One Trypanosoma brucei rhodesiense infection was confirmed; he also had renal and liver failure, pancytopenia and heart block. He was treated with intravenous suramin. He recovered fully over the next 5 months. RECOMMENDATIONS: Medical officers deploying to eastern and southeastern Africa must be familiar with the common presenting signs and symptoms of T b rhodesiense sleeping sickness, and should have access to a reliable local microscopy service at all times. Confirmed sleeping sickness requires immediate transfer to a tertiary diagnostic and treatment centre, where suramin (for T b rhodesiense infection) or pentamidine (for T b gambiense) and also melarsoprol (for Stage Two disease) must be immediately available.

Interaction of cocultured decidual endothelial cells and cytotrophoblasts in preeclampsia.

Disturbed cell-cell communication between trophoblasts and the maternal endothelium may be responsible for the deficient endovascular invasion seen in preeclampsia. In vitro studies have been hampered by lack of suitable models to directly examine interactions between these cell types. Using a bilayer coculture model, we examined the effect of decidual endothelial cells on matrix metalloproteinase secretion and the migration of cytotrophoblasts from preeclamptic pregnancies. Cells were incubated on semipermeable membranes in 20% or 2% O(2) with or without the tumor promoter phorbol 12-myristate 13-acetate, which activates matrix metalloproteinase-2 and -9 in endothelial cells. Cytotrophoblasts from preeclamptic pregnancies secreted significantly less matrix metalloproteinase-2 and -9 than their normal counterparts. Although decidual endothelial cells downregulated cytotrophoblast migration in normal pregnancy, this was not observed in cocultures with cytotrophoblasts from preeclamptic pregnancies. In addition, cytotrophoblasts from preeclamptic pregnancies altered phorbol myristate acetate-induced activation of endothelial matrix metalloproteinases. Hypoxia increased cytotrophoblast migration when cells were incubated alone but not in coculture with decidual endothelial cells due to increased adhesion between the two cell types. These results suggest dysfunctional interactive regulation of migration and matrix metalloproteinase secretion in preeclampsia that could result in abnormal endovascular trophoblast invasion of the maternal vasculature.

A synthesis of dominant ecological processes in intensive shrimp ponds and adjacent coastal environments in NE Australia.

One of the key environmental concerns about shrimp farming is the discharge of waters with high levels of nutrients and suspended solids into adjacent waterways. In this paper we synthesize the results of our multidisciplinary research linking ecological processes in intensive shrimp ponds with their downstream impacts in tidal, mangrove-lined creeks. The incorporation of process measurements and bioindicators, in addition to water quality measurements, improved our understanding of the effect of shrimp farm discharges on the ecological health of the receiving water bodies. Changes in water quality parameters were an oversimplification of the ecological effects of water discharges, and use of key measures including primary production rates, phytoplankton responses to nutrients, community shifts in zooplankton and delta15N ratios in marine plants have the potential to provide more integrated and robust measures. Ultimately, reduction in nutrient discharges is most likely to ensure the future sustainability of the industry.

In vitro migration of cytotrophoblasts through a decidual endothelial cell monolayer: the role of matrix metalloproteinases.

Deficient trophoblast invasion is a major feature of pre-eclampsia. In vitro studies suggest that in normal pregnancy, maternal cells may play a role in controlling trophoblast invasion, although the exact nature of the regulatory interactions between these cells is not fully understood. To examine the effect of maternal-placental cell interactions on matrix metalloproteinase (MMP) secretion and endovascular cytotrophoblast migration in normal pregnancy and in pre-eclampsia, we performed co-culture experiments using cytotrophoblasts from normal pregnancies, together with decidual endothelial cells from both normal and preeclamptic pregnancies. Cells were incubated on semi-permeable membranes with or without phorbol 12-myristate 13-acetate (PMA). Results showed that third trimester cytotrophoblasts are migratory under basal conditions and display a different MMP profile from decidual endothelial cells. Co-culture did not damage either cell type and resulted in reduced latent MMP-9 secretion and reduced cytotrophoblast migration. Although PMA upregulated MMPs in decidual endothelial cells, it had no effect on cytotrophoblast MMP secretion. PMA, however, reduced cytotrophoblast migration. Pre-eclamptic decidual endothelial cells showed reduced MMP-1 secretion, but overall were not different in co-culture from normal endothelial cells. This study demonstrates the effectiveness of a bilayer co-culture model to study maternal-foetal cell interactions and provides evidence that maternal cells may contribute to the control of endovascular cytotrophoblast invasion.

Molecular strain typing of Trichophyton rubrum indicates multiple strain involvement in onychomycosis.

BACKGROUND: Trichophyton rubrum is an important cause of onychomycosis. Molecular strain typing methods have recently been developed to address questions of epidemiology and source of relapse following treatment. OBJECTIVES: To determine whether T. rubrum nail infections are caused by one or more strains of this fungus. METHODS: Nail specimens from 10 patients with onychomycosis due to T. rubrum were cultured and five colonies per culture plate were selected for molecular strain typing. DNA was extracted from these isolates and subjected to a polymerase chain reaction-based typing method that analyses variations in numbers of repetitive elements in the non-transcribed spacer region of the ribosomal RNA gene repeats. RESULTS: In six of 10 specimens, there were two or more T. rubrum strain types present. CONCLUSIONS: This preliminary study suggests that in many cases of fungal nail infection by T. rubrum, multiple strains are involved. This has important implications for epidemiological studies and possibly for therapy.

Analysis of 2beta-carbomethoxy-3beta-(4-fluorophenyl)-N-(3-iodo-E-allyl)nortropane in rat plasma. II. Pharmacokinetic profile in male and female Sprague-Dawley rats evaluated by capillary electrophoresis.

This paper describes a pharmacokinetic study performed in Sprague-Dawley rats after i.v. administration of a single 6-mg/kg dose of 2beta-carbomethoxy-3beta-(4-fluorophenyl)-N-(3-iodo-E-allyl)nortropane (Altropane). Plasma samples were collected from the retro-orbital sinus at times up to 3 h after drug administration, extracted by solid-phase extraction, and the drug levels determined by capillary electrophoresis (CE). Pharmacokinetic parameters were determined by a standard noncompartmental model using WinNonlin version 1.5. The maximum plasma concentrations, clearances of the drug, and areas under the curve for male and female rats were 5.74 and 7.26 microg/ml, 135.7 and 98.5 ml/kg x min, and 44.23 and 60.92 microg x min/ml, respectively. The drug was cleared very rapidly from the systemic circulation, with a terminal t(1/2) of 7 to 10 min and a mean residence time of about 11 min for both sexes. The volume of distribution was approximately 1 l/kg. No metabolites were detected when the samples were analyzed individually. However, after samples were pooled and concentrated, traces of two unknown peaks that may represent metabolites were detected in concentrates from the last two timepoints. Part I of this work [J. Chromatogr. A, 895 (2000) 87] describes validation of CE methods for the analysis of aqueous and plasma samples of Altropane, including its solid-phase extraction from rat plasma.

Human endothelial gelatinases and angiogenesis.

Endothelial cell invasion is an essential event during angiogenesis (formation of new blood vessels). The process involves the degradation of the basement membrane and the underlying interstitium. The matrix metalloproteinase (MMP) family is considered to be primarily responsible for matrix degradation. Two members of the family, gelatinase A and B play an important role in angiogenesis. This review outlines recent findings on their regulation in human endothelial cells. Latent gelatinase B is secreted from endothelial cells. This enzyme can also accumulate in the cytosol as an active enzyme, free of TIMP-1. In contrast, latent gelatinase A is constitutively secreted from the cells. Unlike other MMPs, gelatinase A activation occurs on the cell membrane and is mediated by MT1-MMP. A number of physiological activators have recently been described. These include thrombin and activated protein C, both of which activate gelatinase A independent of the MT1-MMP pathway. These new findings may lead to therapeutic interventions for the treatment of angiogenic-dependent diseases such as cancer and arthritis.