• Hepatic drug targeting: phase I evaluation of polymer-bound doxorubicin.

      Seymour, Leonard W.; Ferry, David R.; Anderson, David; Hesslewood, Stuart; Julyan, Peter J.; Poyner, Richard; Doran, Jayne; Young, Annie M.; Burtles, Sally; Kerr, David J. (American Society of Clinical Oncology, 2002)
      PURPOSE: Preclinical studies have shown good anticancer activity following targeting of a polymer bearing doxorubicin with galactosamine (PK2) to the liver. The present phase I study was devised to determine the toxicity, pharmacokinetic profile, and targeting capability of PK2. PATIENTS AND METHODS: Doxorubicin was linked via a lysosomally degradable tetrapeptide sequence to N-(2-hydroxypropyl)methacrylamide copolymers bearing galactosamine. Targeting, toxicity, and efficacy were evaluated in 31 patients with primary (n = 25) or metastatic (n = 6) liver cancer. Body distribution of the radiolabelled polymer conjugate was assessed using gamma-camera imaging and single-photon emission computed tomography. RESULTS: The polymer was administered by intravenous (i.v.) infusion over 1 hour, repeated every 3 weeks. Dose escalation proceeded from 20 to 160 mg/m(2) (doxorubicin equivalents), the maximum-tolerated dose, which was associated with severe fatigue, grade 4 neutropenia, and grade 3 mucositis. Twenty-four hours after administration, 16.9% +/- 3.9% of the administered dose of doxorubicin targeted to the liver and 3.3% +/- 5.6% of dose was delivered to tumor. Doxorubicin-polymer conjugate without galactosamine showed no targeting. Three hepatoma patients showed partial responses, with one in continuing partial remission 47 months after therapy. CONCLUSION: The recommended PK2 dose is 120 mg/m(2), administered every 3 weeks by IV infusion. Liver-specific doxorubicin delivery is achievable using galactosamine-modified polymers, and targeting is also seen in primary hepatocellular tumors.
    • High Performance liquid chromatography-mass spectrometry analysis for rat metabolism and pharmacokinetic studies of lithospermic acid B from danshen

      Cui, Liang; Chan, Wan; Wu, Jian-Lin; Jiang, Zhi-Hong; Chan, Kelvin C.; Cai, Zongwei (Amsterdam: Elsevier, 2008)
      Metabolism and pharmacokinetic studies on rat were conducted for lithospermic acid B, one of the components from Radix Salviae Miltiorrhizae (danshen) that shows many bioactivities. Liquid chromatography–electrospray ionization mass spectrometry method was applied for the determination of lithospermic acid B and its metabolites in samples from in vitro and in vivo metabolism studies. Rat plasma samples collected after intravenous administration were analyzed for obtaining pharmacokinetic data of lithospermic acid B. Four O-methylated metabolites, namely one monomethyl-, two dimethyl- and one trimethyl-lithospermic acid B, were detected when lithospermic acid B was incubated in rat hepatic cytosol. These four metabolites were also detected in rat bile, plasma and feces samples after intravenous administration of lithospermic acid B. The in vitro and in vivo results indicate that the methylation is the main metabolic pathway of lithospermic acid B. The danshen component and its methylated metabolites were excreted to rat bile and feces. (Elsevier)
    • Human endogenous retrovirus HERV-K10 implicated in rheumatoid arthritis and systemic lupus erythematosus: potential pathological triggers?

      Nelson, Paul N.; Shaw, M.; Roden, Denise A.; Freimanis, Graham L.; Nevill, Alan M.; Rylance, Paul (Czech Republic: Palacky University, Olomouc: Faculty of Medicine and Dentistry, 2006)
      Human endogenous retroviruses (HERVs) are a group of integrated RNA viruses within our human genome. Whilst many are regarded as defective, a number possess the potential to generate retroviral products. Indeed HERVs such as those belonging to the HERV-K family produce retroviral particles in the teratocarcinoma cell line GH and the breast cancer cell line T47D. It has been argued that some retroelements may be beneficial to the human host, perhaps conferring a selective advantage, whereas others may be harmful. Furthermore certain HERVs might be involved in the pathogenesis of autoimmune diseases. The precise mechanisms in diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) may include molecular mimicry and superantigen motifs that evoke and augment unwarranted immune responses. The precise mechanisms in diseases such as rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) may include molecular mimicry and superantigen motifs that evoke and augment unwarranted immune responses. In the RA joint, tissue destruction is evident over time with recruitment of lymphoid and other cells plus the presence of rheumatoid factor that exhibits increased affinity and change in isotype; evidence of an antigen-driven immune response. The precise trigger of course, remains unknown although certain HERVs have been implicated. In a previous study we found evidence for increased expression of HERV-K10 mRNA in patients with RA. Here we have extended this work by investigating the serological expression to HERV-K10 in patients with RA, SLE, osteoarthritis, normals and other inflammatory disease groups. The study utilised a novel peptide ELISA immunoassay using segments of HERV-K10 identified through bioinformatic analysis. In particular, biotinylation of peptides was necessary for serological discrimination between patients. Overall a significant difference (p<0.05) was found for RA patients in terms of antibody activity to HERV-K10. There was also an increased level of antibodies to HERV-K10 in patients with renal lupus although this was below the level of significance. It is possible that HERV-K10 could act as a trigger in RA/SLE through regions of similarity to host proteins. In this case, the immune response to HERV-K10 could lead to collateral damage and pathogenesis of disease.
    • Human endogenous retroviruses: transposable elements with potential?

      Nelson, Paul N.; Hooley, Paul; Roden, Denise A.; Ejtehadi, H. Davari; Rylance, Paul; Warren, Phil; Martin, Jan H.; Murray, Paul G. (Wiley InterScience, 2004)
      Human endogenous retroviruses (HERVs) are a significant component of a wider family of retroelements that constitute part of the human genome. These viruses, perhaps representative of previous exogenous retroviral infection, have been integrated and passed through successive generations within the germ line. The retention of HERVs and isolated elements, such as long-terminal repeats, could have the potential to harm. In this review we describe HERVs within the context of the family of known transposable elements and survey these viruses in terms of superantigens and molecular mimics. It is entirely possible that these mechanisms provide the potential for undesired immune responses.
    • ICAM-1 expression and leukocyte behavior in the microcirculation of chronically ischemic rat skeletal muscles.

      Anderson, Stephen I.; Shiner, Ruth; Brown, Margaret D.; Hudlicka, Olga (Elsevier, 2006)
      In muscle microcirculation, short periods of ischemia followed by reperfusion are known to upregulate leukocyte and endothelial adhesion molecules, but little is known about leukocyte adherence and ICAM-1 expression during chronic ischemia or any likely effect of muscle activity which is recommended in chronic ischemia due to peripheral arterial disease. Leukocyte rolling and stationary adhesion were observed in post-capillary venules in ischemic and contralateral rat extensor digitorum longus (EDL) muscles 3 and 7 days after unilateral ligation of the common iliac artery and in 3-day ischemic EDLs that were electrically stimulated on days 1 and 2 post-ligation (7 x 15 min per day). ICAM-1 was localized immunohistochemically to venular vessels in all muscles. Following ligation, use of the ischemic leg was observed to be restricted for the first 3 days, returning to normal by 7 days. After 3 days, leukocyte rolling/adherence and ICAM-1 expression were no different in ischemic than control muscles, but all were increased in contralateral muscles. In ischemic muscles, electrical stimulation doubled the numbers of rolling leukocytes and upregulated ICAM-1 expression. After 7 days, increased muscle activity as a result of natural movement also resulted in greater ICAM-1 expression, a 4- to 5-fold increase in rolling leukocyte numbers and a 3-fold increase in stationary adherent leukocytes. Chronic ischemia thus increases ICAM-1 and leukocyte adherence in muscle microcirculation only when combined with contractile activity. Post-capillary venular endothelium may be modified by muscle acidosis when contractions are performed under low flow conditions or by changes in rheological (shear force) factors.
    • Identification and biological applications of rhegnylogically-organized cell penetrating peptides.

      Howl, John D.; Jones, Sarah (Australian Peptide Association, 2007)
      Introduction: Many different cell penetrating peptides (CPPs) have been utilized as vectors to affect the highly efficient intracellular delivery of bioactive moieties. A majority of such studies employ sychnologically-organized tandem combinations of a cargo (message) and a CPP (address). To date, bioactive cargoes have included peptides, proteins and a range of oligonucleotides attached either by direct chemical conjugation or as a component of a larger macromolecular complex. Moreover, a majority of CPPs, including the commonly used sequences Tat and penetratin, are designed to be both biologically and toxicologically inert. More recently, a QSAR-based algorithm has been developed to predict cryptic polycationic CPP motifs within the primary sequences of proteins. As described here, this novel technology has enabled the study of rhegnylogic CPPs in which multiple pharmacophores for cellular penetration and desirable biological activities are discontinuously organized within the primary sequence of single peptide. This organization differs from the more commonly utilized sychnologic strategy which joins functionally discrete and continous address and messages together in a tandem construct.
    • Identification of differentially expressed genes in paediatric ependymoma

      Suarez-Merino, Blanca; Hubank, Mike; Hayward, Richard; Harkness, William; Thompson, Dominic; Phipps, Kim; Revesz, Tamas; Darling, John L.; Thomas, David G.; Warr, Tracy (Society for Neuro-Oncology and Duke University Press, 2003)
      To date, the genetic events that contribute to the pathogenesis of ependymoma are essentially unknown. Furthermore, previous cytogenetic studies have demonstrated that approximately 50% of tumors have no detectable chromosome abnormalities. In this study, we used the Affymetrix GeneChip U95Av2 microarrays to generate gene expression profiles in 11 pediatric ependymoma, comprising 6 fresh frozen samples and 5 short-term cultures. A total of 107 genes were differentially expressed in both the biopsies and cell cultures compared with normal control tissue derived from corpus callosum. The 84 genes with increased expression included many encoding adhesion and extracellular matrix proteins; genes involved in the cell cycle, such as cyclin D1, CDK2, CDK4, and Wee1; transcription factors such as Zic1; and known oncogenes such as c-myc, WNT5A, JUN, TC21, RAB36, and FOP. An interesting group also found to be overexpressed comprises the insulin-like growth factor binding proteins IGFBP2, IGFBP3, and IGFBP4, which may be responsible for the autostimulation of cell growth in these tumors. Of the 23 genes that were underexpressed by 5-fold or less, 6 were of unknown function. Others included the apoptotic control gene ATIP the DAAM1 gene associated with the Wnt/frz oncogenic pathway, and genes involved in vesicle trafficking and recycling within and across cells, such as NPC1, RAB40B, TJP2, and SH3GL3. We have identified a number of candidate genes and genetic pathways that have not previously been associated with the pathogenesis of pediatric ependymoma. Further evaluation of the expression or mutation status of these genes will elucidate their roles in ependymoma development.
    • Identification of extensive genomic loss and gain by comparative genomic hybridisation in malignant astrocytoma in children and young adults.

      Warr, Tracy; Ward, Samantha; Burrows, J.; Harding, Brian; Wilkins, Peter; Harkness, William; Hayward, Richard; Darling, John L.; Thomas, David G. (Wiley Interscience, 2001)
      Although astrocytomas are the most common central nervous system tumours in all age groups, there is substantial evidence that tumours arising in young patients (< 25 years of age) do not have the same genetic abnormalities that are characteristic of tumours in older patients. Furthermore, novel, consistent changes have not been identified in astrocytomas in children and young adults. We analysed 13 malignant astrocytomas from young patients using comparative genomic hybridisation. Regions of genomic imbalance were identified in 10 cases. The most common recurrent copy number aberrations were loss of 16p (54% of cases), 17p (38%), 19p (38%), and 22 (38%) and gain on 2q (38%), 12q (38%), 13 (38%), 4q (31%), 5q (31%), and 8q (31%). Seven regions of high copy number amplification were observed at 8q21-22 (three cases), 7q22-23 (two cases), and 1p21-22, 2q22, 12q13-pter, 12q15-21, and 13q11-14 (one case each). This study provides evidence of new characteristic chromosomal imbalances from which potential candidate genes involved in the development of malignant astrocytoma in children and young adults may be identified.
    • Identification of the pocket factors in a picornavirus

      Smyth, M.S.; Martin, Jan H.; Pettitt, T.; Symonds, A. (Springer Wien, 2003)
      Bovine enterovirus (BEV), along with other enteroviruses and the rhinoviruses, has a hydrophobic pocket within structural protein VP1. In the crystal structures of these viruses there is electron density commensurate with a non-protein molecule within the pocket. These molecules, termed pocket factors, have been shown to stabilise the capsid and their removal from the pocket is a necessary prerequisite to uncoating. The pocket factors have been proposed, from the electron densities and uncoating studies, to be short chain fatty acids. In order to identify the pocket factor of BEV, we have grown and purified the virus in an identical manner to that used for the crystal structure determination and have performed a lipophilic extraction. Palmitic acid, C16:0, was the most abundant accounting for 40.8% by mass of the lipophilic extract (39.3 mol%). Myristic acid C14:0, was next most abundant at 18.5% by mass (20.0 mol%). In addition, we have identified other fatty acids in smaller proportions. We have therefore shown that BEV contains saturated fatty acid pocket factors of varying chain length. We have also compared the profile of the fatty acyl chain composition of BEV with those for uninfected BHK-21 cell plasma membrane and endoplasmic reticulum extracts.
    • In vitro culture of malignant brain tumours

      Darling, John L. (New York: John Wiley, 2003)
      THIS BOOK: This comprehensive resource provides stepwise protocols for the in vitro cultivation of the major types of human tumors. Written in a reader-friendly style, this book is organized by specific tumor, discussing cultures for lung, gastric, colorectal, pancreas, bladder, prostate, ovary, cervix, mammary carcinoma, myoepithelium, squamous, melanoma, lymphoma, glioma, and neuroendocrine tumors. Detailed techniques for initiation, propagation, and characterization of established cell lines are presented. Drug treatment, selection, differentiation, assays for malignant cells, risks, and applications are discussed. An extensive listing of vendors for equipment and other cell culture products is also included. (Wiley)
    • In vitro evaluation of cancer-specific NF-kappaB-CEA enhancer-promoter system for 5-fluorouracil prodrug gene therapy in colon cancer cell lines.

      Guo, X.; Evans, T.R.J.; Somanath, S.; Armesilla, Angel Luis; Darling, John L.; Schatzlein, A.; Cassidy, James; Wang, Weiguang (Nature Publishing Group, 2007)
      Nuclear factor-kappa B (NF-kappaB) is a transcription factor with high transcriptional activity in cancer cells. In this study, we developed a novel enhancer-promoter system, kappaB4-CEA205, in which the basal carcinoembryonic antigen (CEA) promoter sequence (CEA205) was placed downstream of the four tandem-linked NF-kappaB DNA-binding sites (kappaB4). In combination with a kappaB4 enhancer, the transcriptional activity of the CEA promoter was significantly enhanced (three- to eight-fold) in cancer cell lines but not in normal cells. In cancer cell lines, the transcriptional activity of kappaB4-CEA205 was comparable with that of the SV40 promoter. We also constructed vectors in which the thymidine phosphorylase (TP) cDNA was under the control of CEA205, kappaB4, kappaB4-CEA205 and CMV promoters, respectively. TP protein and enzyme activity were detected at comparable levels in kappaB4-CEA205- and CMV-driven TP cDNA-transfected cancer cell lines (H630 and RKO). The kappaB4-TP and CEA205-TP-transfected cell lines, respectively, only demonstrated negligible and low levels of TP protein and enzyme activity. Both CMV- and kappaB4-CEA205-driven TP cDNA transiently transfected cells were 8- to 10-fold sensitised to 5-fluorouracil (5-FU) prodrug, 5'-deoxy-5-fluorouradine (5'-DFUR), in contrast to only 1.5- to 2-fold sensitised by the kappaB4- and CEA205-driven TP cDNA-transfected cells. The bystander killing effect of CMV- and kappaB4-CEA205-driven TP cDNA-transfected cells was comparable. This is the first report that indicates that the NF-kappaB DNA-binding site could be used as a novel cancer-specific enhancer to improve cancer-specific promoter activity in gene-directed enzyme prodrug therapy.
    • Increased non-transferrin bound iron in plasma-depleted SAG-M red blood cell units.

      Marwah, S.S.; Blann, A.D.; Harrison, P.; Lumley, M.A.; Wright, J.; McDowell, J.; Phillips, Jonathan D.; Rea, C.; Bareford, D. (Wiley InterScience, 2002)
      BACKGROUND AND OBJECTIVES: Non-transferrin bound iron (NTBI) is associated with increased morbidity in a number of transfusion-dependent disease states such as the severe haemoglobinopathies. We hypothesized that this may be related to excess NTBI present in plasma-depleted red blood cell units that are free of clear haemolysis. MATERIALS AND METHODS: The level of NTBI was determined using the bleomycin assay in samples from 20 stored plasma-depleted red cell units, at approximate 5-day intervals up to day 33 after donation. Forty units of fresh-frozen plasma (FFP) and 40 units of platelet concentrates were used as negative controls, and samples from 12 units of FFP were also serially assessed. RESULTS: Median [interquartile range (IQR)] NTBI was 0 microm (0-0.35) in samples taken from units 3-10 days after donation. Thereafter, the levels of NTBI increased, becoming significant (median 3.05; IQR: 0.05-6.7 microm) 17-22 days after donation. After 30 days, NTBI was detectable in all red cell units. NTBI was undetectable in platelet concentrates and FFP. CONCLUSIONS: Increased levels of NTBI become detectable 17-22 days after donation and increase further with storage time. This excess NTBI may promote bacterial infection in iron-loaded individuals.
    • Individualised assessment of response to clopidogrel in patients presenting with acute coronary syndromes: a role for short thrombelastography?

      Cotton, James M.; Worrall, A. M.; Hobson, A. R.; Smallwood, A.; Amoah, V.; Dunmore, Simon J.; Nevill, Alan M.; Raghuraman, R. P.; Vickers, J.; Curzen, N. (2010)
      INTRODUCTION: There is considerable interindividual variation in response to the antiplatelet agent clopidogrel. Hyporesponse predicts negative outcomes in patients presenting with a variety of ischemic cardiac conditions and following intracoronary stent placement. Many tests of clopidogrel activity are time consuming and complex. Short thromboelastography (s-TEG) allows rapid measurement of platelet clopidogrel response. AIMS: We initiated this study to investigate the utility of s-TEG in assessing the response to clopidogrel in patients presenting with acute coronary syndromes (ACS) and to compare these results with established clopidogrel monitoring techniques. METHODS: Patients admitted with unstable angina (UA) or Non ST elevation myocardial infarction (NSTEMI) undergoing coronary angiography were recruited. After routine loading with clopidogrel, all patients were tested with s-TEG and Accumetrics Verify-Now rapid platelet function analyzer (VN-RPFA). We used the modified TEG technique of measuring area under the curve at 15 min (AUC15), which allows a rapid estimation of antiplatelet response. Vasodilator-stimulated phosphoprotein phosphorylation (VASP) was also tested in a subgroup of patients. Clinical follow-up was obtained at 1 year. s-TEG results were correlated with VN-RPFA and VASP findings. RESULTS: A total of 49 patients (33 male, mean age 63) were recruited and tested with s-TEG and VN-RPFA and a total of 39 patients were also assessed with VASP. s-TEG readings correlated well with VN-RPFA (r(2)= 0.54, P < 0.0001) and VASP (r(2)= 0.26, P= 0.001). CONCLUSION: s-TEG provides timely results which compare to current tests of clopidogrel activity. This technique can also be used to measure a variety of other clotting parameters and as such could develop into a valuable near patient test for the interventional cardiologist.
    • Influence of co-administrated sinomenine on pharmacokinetic fate of paeoniflorin in unrestrained conscious rats.

      Liu, Zhong Qiu; Zhou, Hua; Liu, Liang; Jiang, Zhi-Hong; Wong, Yuen Fan; Xie, Ying; Cai, Xiong; Xu, Hong-Xi; Chan, Kelvin C. (Elsevier, 2005)
      Paeonia lactiflora Pall. (Ranunculaceae) root and Sinomenium acutum Rehder and Wilson (Menispermaceae) stem are two herbs widely used in Chinese medicine to treat rheumatoid arthritis. While, in theory, either herb could be used alone, in practice, Chinese medicine practitioners prescribe them together. Studies on pharmacokinetic interaction between the active constituents of these two herbs (paeoniflorin and sinomenine, respectively) provide empirical evidence to support their clinical practice. A single dose of paeoniflorin (150 mg/kg) alone and with sinomenine hydrochloride (90 mg/kg) was administered by gastric gavage to unrestrained conscious male Sprague-Dawley rats (n=6, 250-300 g). Blood samples were collected periodically via a jugular vein before and after dosing from 10 min to 12 h. A high-performance liquid chromatographic (HPLC) assay was developed to determine the plasma concentrations of paeoniflorin. Non-compartmental pharmacokinetic profiles were constructed by using the software PK Solutions 2.0. The pharmacokinetic parameters were compared using unpaired Student t-test. After co-administration of sinomenine, the peak plasma concentration of paeoniflorin was elevated (P<0.01), the peak time was delayed (P<0.01), the AUC(0-t) was increased (P<0.001), the mean residence time (MRT) was prolonged (P<0.01), the C(L) was decreased (P<0.01) and the V(d) was reduced (P<0.05). These results indicate that sinomenine hydrochloride at 90 mg/kg significantly improved the bioavailability of paeoniflorin in rats.
    • Influence of lactose carrier particle size on the aerosol performance of budesonide from a dry powder inhaler

      Kaialy, Waseem; Alhalaweh, Amjad; Velaga, Sitaram P.; Nokhodchi, Ali (Elsevier, 2012-09)
      The purpose of this study was to evaluate the effect of carrier particle size on properties of dry powder and its effect on dry powder inhaler (DPI) performance. Commercial α-lactose-monohydrate, a commonly used carrier in DPI formulations, was carefully sieved to obtain different lactose size fractions, namely Lac A (90–125 μm), Lac B (63–90 μm), Lac C (45–63 μm), Lac D (20–45 μm), and Lac E (< 20 μm). The lactose samples were analysed in terms of size, shape, solid state, density, and flowability. Lactose particles were blended with budesonide (< 5 μm) powder to generate five different formulations. These formulations were then evaluated in terms of budesonide–lactose adhesion properties, drug content homogeneity, and in vitro aerosolisation performance. The results demonstrated that lactose samples with smaller particle volume mean diameter have higher amorphous lactose content, higher true density (linear, r2 = 0.9932), higher surface smoothness (linear, r2 = 0.8752), smaller angularity (linear, r2 = 0.921), smaller bulk density, higher porosity (linear, r2 = 0.914), poorer flowability, and higher specific surface area. In general, the smaller the lactose particles the smaller are the budesonide–lactose adhesion properties. Budesonide formulated with smaller lactose particles exhibited smaller aerodynamic diameter and higher amounts of budesonide were delivered to lower stages of the impactor indicating improved DPI aerosolisation performance. However, the use of lactose particles with smaller volume mean diameter had a detrimental effect on budesonide content homogeneity and caused an increase in the amounts of budesonide deposited on oropharyngeal region. Therefore, particle size of the lactose within dry powder inhaler formulations should be selected carefully. Accordingly, higher drug aerosolisation efficiency of lactose particles with smaller size may have to be balanced due to considerations of other disadvantages including poorer flowability, reduced formulation stability, higher potential side effects, and higher dose variability.
    • Inhibition of prostaglandin synthesis does not alter the decrease in pre-capillary resistance in the human calf in response to small cumulative increases in venous congestion

      Anderson, Stephen I.; Brown, Margaret D. (Portland Press, 2005)
      The decrease in pre-capillary resistance in the human calf during gradual cumulative increases in venous congestion pressure has been proposed to represent vasodilator signalling between the venous and arterial microcirculations. The present study investigated whether prostaglandins are involved in this local flow regulation by measuring calf blood flow and microvascular filtration capacity using strain gauge plethysmography in young male subjects before (baseline) and after taking either ibuprofen, an inhibitor of prostaglandin synthesis (1600 mg over 2 days), or placebo. At baseline, inflation of a thigh cuff to 50 mmHg in steps of 10 mmHg, each held for 5 min, did not decrease arterial inflow, confirming a reduction of pre-capillary resistance. Ibuprofen reduced resting calf blood flow by 35% (P<0.001), but flow at a Pcuff (cuff pressure) of 50 mmHg was 97% of this value, i.e. pre-capillary resistance had decreased to the same extent as before inhibition of prostaglandin synthesis. Ibuprofen also reduced microvascular filtration capacity (2.98±1.20 compared with 3.71±0.89 ml·min-1·100 ml-1·mmHg-1×10-3; P<0.05), probably due to a combination of reduced arterial inflow and lower venous pressure (8.5±5.2 compared with 12.6±2.8 mmHg; P<0.05) that moderated capillary hydrostatic pressure to override direct effects of inhibition of prostaglandin synthesis on permeability. Placebo was without effect on any measurement. It is unlikely therefore that prostaglandin-mediated vasodilator signals, which have been demonstrated between paired veins and arteries, are important in local vasodilation in response to venous congestion.
    • Inorganic polyphosphate in Dictyostelium discoideum: influence on development, sporulation, and predation.

      Zhang, Haiyu; Gómez-García, Maria R.; Brown, Michael R. W.; Kornberg, Arthur (National Academy of Sciences, 2005)
      Dictyostelium discoideum, a social slime mold that forms fruiting bodies with spores, depends on inorganic polyphosphate (poly P) for its cycles of development and for nutritional predation on bacteria. The synthesis of poly P, a polymer of tens or hundreds of phosphate residues linked by high energy, ATP-like bonds, is catalyzed in most bacteria by poly P kinase (PPK1). The eukaryote D. discoideum possesses a homolog of PPK1. We report here that mutants of D. discoideum PPK1 (DdPPK1) have reduced levels of poly P and are deficient in development. Fruiting bodies are smaller and produce fewer spores, which appear to germinate like the wild type (WT). The DdPPK1 mutant formed smaller plaques on bacterial lawns compared with those of the WT. Predation by D. discoideum, assessed by uptake and digestion of Klebsiella aerogenes, showed that fewer bacteria were taken up by the DdPPK1 mutant compared with the WT and were killed less rapidly, indicating a role of poly P and/or DdPPK1 in phagocytosis. On Pseudomonas aeruginosa lawns, cleared plaques were observed with the bacterial PPK1 mutant but not with the WT P. aeruginosa. Thus, poly P is important in predation both for the predator and prey.
    • Inorganic polyphosphate in the origin and survival of species.

      Brown, Michael R. W.; Kornberg, Arthur (National Academy of Sciences, 2004)
      Inorganic polyphosphate (poly P), in chains of tens to hundreds of phosphate residues, linked by high-energy bonds, is environmentally ubiquitous and abundant. In prebiotic evolution it could have provided a flexible, polyanionic scaffold to assemble macromolecules. It has been conserved in every cell in nature. In prokaryotes, a major poly P synthetic enzyme is poly P kinase 1 (PPK1), which is found in 100 bacterial genomes, including numerous pathogens. Null mutants of PPK1, with low poly P levels, are defective in survival: namely, they show defective responses to physical/chemical stresses and predation. Pathogens with a PPK1 deletion are defective in biofilm formation, quorum sensing, general stress and stringent responses, motility, and other virulence properties. With the exception of Dictyostelium, PPK1 is absent in eukaryotes and provides a novel target for chemotherapy that would affect both virulence and susceptibility to antibacterial compounds. Remarkably, another PPK in Dictyostelium discoideum (PPK2) is an actin-related protein (Arp) complex that is polymerized into an actin-like filament, concurrent with its reversible synthesis of a poly P chain from ATP.
    • Interactions of cell penetrating peptide Tat with model membranes: a biophysical study.

      Dennison, Sarah R.; Baker, Rachael D.; Nicholl, Iain D.; Phoenix, David A. (Elsevier Science Direct, 2007)
      The protein transduction domain of the HIV-1 transactivator of transcription, Tat (Tat((48-60))), has been shown to transport P10, a cytotoxic peptide mimic of the cyclin dependent kinase inhibitor p21WAF1/CIP1, into the nucleus of cancerous cells and induce apoptosis. Here, monolayer studies were used to investigate the membrane interactions of Tat((48-60)), P10 and the construct Tat((48-60))P10. It was found that Tat((48-60)) showed no significant surface activity but that both P10 and Tat((48-60))P10, were highly surface active, inducing surface pressure changes of 9.7 and 8.9mNm(-1), respectively, with DMPS monolayers. The comparison of Tat((48-60))P10 and P10 surface interactions would be consistent with a hypothesis that the cargo attachment influences the capacity of the Tat-protein transduction domain to mediate transport across membranes either directly or via localisation of the construct at the membrane interface.