Inhibitory Principle Research Articles

BARC Biostimulant 1, a novel Streptomyces rochei C3-based formulation for disease biocontrol and plant growth promotion

Pythium aphanidermatum is a very destructive plant pathogen causing damping-off disease of vegetable seedlings in nurseries. In addition, this plant pathogenic oomycete causes seed, stem and rhizome rot, as well as post-harvest diseases. Currently, the disease management relies heavily on high dosages of chemicals like metalaxyl and copper oxychloride biocides, which are not only expensive, but are hazardous to the ecosystem. In our quest for developing a disease management strategy that is economical and safe, we isolated a strain of an actinobacterium that is highly inhibitory to P. aphanidermatum, the major cause of damping-off disease. Based on morphology and 16S rRNA sequence analysis, we identified this isolate as Streptomyces rochei. Using an LC-MS/MS analysis, the inhibitory principle has been tentatively identified as (4 S)-4,5-dihydro-4-hydroxygeldanamycin.We developed a novel formulation “BARC Biostimulant 1” using split tamarind seeds as a substrate and talcum powder as a carrier and S. rochei as the active ingredient. In P. aphanidermatum-infested soil, seed treatment and drenching resulted in about 50 % improvement in germination in greenhouse assay and protected field experiments. Under open field conditions, the germination improved to the extent of 20 %. In addition to suppression of the disease, the formulation also improved plant growth parameters like weight and height of seedlings, and induced defense responses. We further undertook multi-location replicated field trials to evaluate this purely organic formulation as a biostimulant across four states of India. Seed treatment with BARC Biostimulant 1 at the rate of 5 g/ kg seeds enhanced germination of seedlings by 80 % and enhanced fresh weight by 105 % (average of four locations), indicating a good commercial potential of this formulation.

Identification of a highly efficient dual type I/II FMS-like tyrosine kinase inhibitor that disrupts the growth of leukemic cells

Internal tandem duplications (ITDs) in the FMS-like tyrosine kinase-3 (FLT3) are causally linked to acute myeloid leukemia (AML) with poor prognosis. Available FLT3 inhibitors (FLT3i) preferentially target inactive or active conformations of FLT3. Moreover, they co-target kinases for normal hematopoiesis, are vulnerable to therapy-associated tyrosine kinase domain (TKD) FLT3 mutants, or lack low nanomolar activity. We show that the tyrosine kinase inhibitor marbotinib suppresses the phosphorylation of FLT3-ITD and the growth of permanent and primary AML cells with FLT3-ITD. This also applies to leukemic cells carrying FLT3-ITD/TKD mutants that confer resistance to clinically used FLT3i. Marbotinib shows high selectivity for FLT3 and alters signaling, reminiscent of genetic elimination of FLT3-ITD. Molecular docking shows that marbotinib fits in opposite orientations into inactive and active conformations of FLT3. The water-soluble marbotinib-carbamate significantly prolongs survival of mice with FLT3-driven leukemia. Marbotinib is a nanomolar next-generation FLT3i that represents a hybrid inhibitory principle.

An osteopontin-derived peptide inhibits human hair growth at least in part by decreasing fibroblast growth factor-7 production in outer root sheath keratinocytes.

Given that unwanted hair growth (hirsutism, hypertrichosis) can cause major psychological distress, new pharmacological treatment strategies with safe and effective hair growth inhibitors that do not destroy the hair follicle (HF) and its stem cells need to be developed. To establish if osteopontin-derived fragments may modulate human hair growth given that human HFs express the multifunctional, immunomodulatory glycoprotein, osteopontin. Our hypothesis was tested exvivo and invivo by using a newly generated, toxicologically well-characterized, modified osteopontin-derived peptide (FOL-005), which binds to the HF. In organ-cultured human HFs and scalp skin, and in human scalp skin xenotransplants onto SCID mice, FOL-005 treatment (60 nmol L-1 to 3 μmol L-1 ) significantly promoted premature catagen development without reducing the number of keratin 15-positive HF stem cells or showing signs of drug toxicity. Genome-wide DNA microarray, quantitative reverse-transcriptase polymerase chain reaction and immunohistochemistry revealed decreased expression of the hair growth promoter, fibroblast growth factor-7 (FGF7) by FOL-005, while cotreatment of HFs with recombinant FGF7 partially abrogated FOL-005-induced catagen promotion. With caveats in mind, our study identifies this osteopontin-derived peptide as an effective, novel inhibitory principle for human hair growth exvivo and invivo, which deserves systematic clinical testing in hirsutism and hypertrichosis. What's already known about this topic? The treatment of unwanted hair growth (hypertrichosis, hirsutism) lacks pharmacological intervention, with only few and often unsatisfactory treatments available. Osteopontin is prominently expressed in human HFs and has been reported to be elevated during catagen in the murine hair cycle. What does this study add? We tested the effects on hair growth of a novel, osteopontin-derived fragment (FOL-005) exvivo and invivo. In human hair follicles, high-dose FOL-005 significantly reduces hair growth both exvivo and invivo. What is the translational message? High-dose FOL-005 may provide a new therapeutic opportunity as a treatment for unwanted hair growth.

Impact of eyestalk ablation on androgenic gland activity in the freshwater crab Travancoriana schirnerae Bott, 1969 (Decapoda: Gecarcinucidae)

The current study investigated the impact of eyestalk ablation on androgenic gland activity in the freshwater crab Travancoriana schirnerae Bott, 1969 (Decapoda: Gecarcinucidae). Bilateral eyestalk ablation during active phase (March-June) induced hypertrophy and hyperplasia of the gland as evidenced by increased width of the gland, thickness of the connective tissue layer, increase in cell and nuclear diameter with predominance of type I cells. This hypertrophy and hyperplasia was not followed by secretory activity during active phase which possibly suggests the non-functional nature of hypertrophy and hyperplasia. Unilateral destalkation during active phase neither caused hypertrophy nor hyperplasia of the gland cells probably because these crabs may still receive the inhibitory principle from the remaining intact eyestalk causing the gland to remain analogous to that of the unablated controls. The androgenic gland of control crabs during revival phase (January-February) was discerned with a few patches of proliferated type I cells scattered among vacuoles and degenerating cells. The most prominent feature of the gland of bilaterally ablated crabs during revival phase was the extensive proliferation of type I cells. Not much significant change was observed in the morphology and histology of the gland of unilaterally ablated crabs of revival phase.

CpG oligonucleotides as adjuvant in therapeutic vaccines against parasitic infections

Immunostimulatory CpG DNA is recognized by cells of the innate immune system through Toll-like receptor 9 (TLR9). Synthetic CpG oligonucleotides (CpG-ODN) belong to the most potent vaccine adjuvants known today. This is due to their capacity not only to stimulate cells of the innate immune system but also to trigger effectively specific T- and B-lymphocyte responses. This unique quality seems to be superior in the induction of long-term effects and immunological memory. In addition to prophylactic vaccination, we showed that mice already infected with Toxoplasma gondii could be therapeutically vaccinated by the combined use of CpG-ODN and a bradyzoite antigen (BAG1 protein subunit). This treatment was effective against the infection and resulted in a long-term survival of the mice and reduced parasite burden in the brain. Different routes of CpG-ODN vaccine application including intranasal, oral or intraperitoneal delivery were compared with the classical subcutaneous application in two established experimental infection models of murine leishmaniasis and toxoplasmosis. Comparable effects were demonstrated for these modes of inoculation except for the oral uptake of uncoupled CpG-ODN, which resulted in a complete failure of treatment. Detailed studies were performed to optimize the time point of CpG-ODN application. The best results were obtained when the ODN were given within a few days around the infection, in contrast to former trials showing a time window of several weeks for significant oligonucleotide effects in non-infectious models. As CPG-ODN is a synthetic compound, it is not only available in high purity and reproducible quality, but can also be produced with different backbone modifications and sequence modifications. Combination of these possibilities resulted in new molecules that were highly effective as adjuvant in parasite infection models. Finally, our studies revealed not only that bacterial DNA is an effective vaccine adjuvants, but also that Leishmania DNA itself had immunostimulatory properties which are counteracted by a yet undefined inhibitory principle from living Leishmania.

Analysis of frankincense from various Boswellia species with inhibitory activity on human drug metabolising cytochrome P450 enzymes using liquid chromatography mass spectrometry after automated on-line extraction

In our search for herbal remedies with inhibitory activity on cytochrome P450 (CYP) enzymes, we identified extracts of the gum-resin of Boswellia carteri, Boswellia frereana, Boswellia sacra and Boswellia serrata as equally potent, non-selective inhibitors of the major drug metabolising CYP enzymes 1A2/2C8/2C9/2C19/2D6 and 3A4. LC/LC/ESI-MS fingerprint analyses of the boswellic acids 11-keto-β-boswellic acid, α-boswellic acid, β-boswellic acid and their 3- O-acylated derivatives were used for the authentication of the commercially obtained frankincense samples. Although the boswellic acids could be identified as moderate to potent inhibitors of the applied CYP enzymes, they are not the major CYP inhibitory principle of frankincense.

Inhibitory activity of tryptanthrin on prostaglandin and leukotriene synthesis.

The indolo[2,1- b]quinazoline alkaloid tryptanthrin has previously been identified as the cyclooxygenase-2 (COX-2) inhibitory principle in the extract ZE550 prepared from the medicinal plant Isatis tinctoria (Brassicaceae). We here investigated the potential inhibitory activity of tryptanthrin and ZE550 on COX-2, COX-1 in cellular and cell-free systems. A certain degree of selectivity towards COX-2 was observed when COX-1-dependent formation of thromboxane B(2) (TxB(2)) in HEL cells and COX-2-dependent formation of 6-ketoprostaglandin F(1alpha) (6-keto-PGF(1alpha)) in Mono Mac 6 and RAW 264.7 cells were compared. Preferential inhibition of COX-2 by two orders of magnitude was found in phorbol myristate acetate (PMA) activated bovine aortic coronary endothelial cells (BAECs). Assays with purified COX isoenzymes from sheep confirmed the high selectivity towards COX-2. The leukotriene B(4) (LTB(4)) release from calcium ionophore-stimulated human granulocytes (neutrophils) was used as a model to determine 5-lipoxygenase (5-LOX) activity. Tryptanthrin and the extract ZE550 inhibited LTB(4) release in a dose dependent manner and with a potency comparable to that of the clinically used 5-LOX inhibitor zileuton.

Isatis tinctoria – From the rediscovery of an ancient medicinal plant towards a novel anti-inflammatory phytopharmaceutical

An account on the reinvestigation of the old dye and medicinal plant Isatis tinctoria as an anti-inflammatory and current research on the active principles in woad is given. In a broad-based screening, a dichloromethane extract from the leaves displayed significant activities on several clinically relevant targets of inflammation. The cyclooxygenase-2 inhibitory principle was identified with the aid of HPLC-based activity profiling as the alkaloid tryptanthrin. In cell based assays, tryptanthrin strongly inhibited eicosanoid synthesis catalyzed by cyclooxygenase-2 and 5-lipoxygenase. A supercritical carbon dioxide extraction process was developed to replace the dichloromethane extract. Dichloromethane and carbon dioxide extracts showed in vivo anti-inflammatory activity in topical and oral application. With the aid of electrospray ionization liquid chromatography-mass spectrometry coupled skin microdialysis, tryptanthrin was found to penetrate the skin. The penetration from the extract was better than for the pure alkaloid. A screening of 67 woad samples of different geographic origin revealed up to 30-fold differences in tryptanthrin content in leaves.

Serum is a rich source of ligands for the scavenger receptor of hepatic sinusoidal endothelial cells.

The present study was prompted by findings in our laboratory showing that serum effectively inhibits scavenger receptor (SR)-mediated endocytosis in hepatic sinusoidal endothelial cells (SEC). Experiments with SEC in vitro showed that the presence of 20% human serum inhibited endocytosis of SR ligands, 125I-formaldehyde treated bovine serum albumin (FSA) and 125I-nidogen, by 30 and 50%, respectively, whereas pre-heated foetal bovine serum (10%) inhibited endocytosis of 125I-FSA by as much as 56%. Human, bovine and rat serum had similar inhibitory effect on endocytosis in SEC. Fractionation of foetal bovine and human serum on anion exchange chromatography demonstrated that the inhibitory principle co-purified with macromolecules of high negative charge. The serum fraction that most effectively inhibited SR-mediated endocytosis of 125I-FSA did not affect mannose receptor-mediated endocytosis of 125I-mannan to the same extent. Trap-labelled negatively charged serum fraction administered intravenously to rats was eliminated almost exclusively by liver, with a blood decay of 50% over the first 3 min after injection. Isolation of liver cells showed that the populations of Kupffer cells and SEC contained 39 and 61% of liver radioactivity 30 min after injection of trap-labelled negatively charged fractions prepared from pre-heated ('complement inactivated') foetal bovine sera. These findings suggest that the process of serum formation from native blood generates appreciable amounts of macromolecules that compete specifically with the SR for endocytosis in SEC. The inhibitory power of pre-heated serum is particularly great. For this reason pre-heated serum should be used with caution in studies of SR in SEC.

PURIFICATION AND IDENTIFICATION OF A HUMAN DERMAL EXTRACT COMPONENT INHIBITORY TO FIBROBLAST PROLIFERATION

It was previously shown that a citric acid buffer extract of human dermis (extract D) inhibited growth of human diploid fibroblasts in monolayer culture (Muir et al., 1997). Further fractionation has shown that the active principle is probably a proteoglycan, and that retention of its inhibitory activity is dependent on the use protease inhibitors throughout the extraction procedure. Elution of extract D from a DEAE-cellulose column produced four major peaks, each of which was subjected to SDS-PAGE as well as being tested for inhibitory activity on the growth of fibroblasts in culture. Peaks III and IV had no inhibitory effect, but peak I contained highly active material. Gels of this peak showed prominent bands of 120kDa (corresponding to dermatan sulphate proteoglycan II, DS-PG II) and at 45kDa (corresponding to the core protein). The latter band became more prominent when extract D which had been treated with chrondroitinase ABC was electrophoresed. Their identities were verified by Western blotting. Peak II also contained some slower-acting inhibitory material which has as yet to be identified, but contains little or no protein corresponding to the decorin core-protein. The data indicate that the intact decorin molecule, DS-PG II, is the main inhibitory principle in human skin.

Diamine extension of glutaraldehyde crosslinks mitigates bioprosthetic aortic wall calcification in the sheep model.

We previously have been able to show that fixation at increasing concentrations of glutaraldehyde (GA) leads to mitigated rather than facilitated tissue calcification. The purpose of the present study was to introduce additional crosslinks and provide evidence that crosslink density may be an underlying inhibitory principle. Entire aortic roots were chosen to verify the concept on the challenging aortic wall tissue. Porcine aortic roots were crosslinked with 0.2% GA, 3%GA, and 3% GA containing an interim step that introduced diamine bridges. Crosslink efficiency was determined on the basis of shrinkage temperature (SrT degrees ), resistance to protease digestion (RPD), residual amine analysis (RA), and tensile modulus (E(10)). Calcium levels, calcification patterns, and inflammation were assessed after 6 and 24 weeks of implantation in a sheep circulatory model. Crosslink efficiency in aortic wall tissue was moderately affected by increasing the fixative concentration from 0.2% GA to 3% GA (SrT degrees from 85.7 degrees +/- 0.3 degrees to 87.5 degrees +/- 0.3 degrees C, p < 0.002; RPD from 24.2 +/- 1.2 to 29.1 +/- 0.7%, p < 0.003; RA from 0.069 +/- 0.004 to 0.058 +/- 0.003 micromol/mg, p < 0.03, and E(10) from 1.9 +/- 0.11 to 2.94 +/- 0.34 MPa, p < 0.01), but it was distinctly enhanced when diamine bridges were introduced (SrT degrees from 87.5 degrees +/- 0.3 degrees to 93.4 degrees +/- 0.3 degrees C, p << 0.0001; RPD from 29.1 +/- 0.7 to 68.4 +/- 1.8%, p << 0.0001; and E(10) from 2.94 +/- 0.34 to 6.80 +/- 0.61 MPa, p < 0.0003). Aortic wall calcification was reduced significantly by increasing the GA concentration from 0.2 to 3% [37.8%, p = 0.076 (6 weeks) and 34.0%, p = 0.008 (24 weeks)] and further reduced by the introduction of additional diamine [84.0%, p = 0.006 (6 weeks) and 29.8%, p = 0.037 (24 weeks)]. The combined effect of increased GA concentration plus an interim diamine step on aortic wall tissue resulted in a 90% and 53.7% reduction of calcification after 6 weeks and 24 weeks, respectively. The correlation coefficients between calcification and SrT degrees, RDP, and E(10) was -0.9767, -0.9460, and -0.9740, respectively (6 weeks). The inflammatory host reaction regularly found in 0.2% fixed tissue was practically abolished through the introduction of diamine bridges. Our study demonstrated a distinct correlation between the mitigation of aortic wall calcification and three parameters used to assess crosslink density.

Identification and isolation of the cyclooxygenase-2 inhibitory principle in Isatis tinctoria.

Various extracts prepared from the traditional dye and medicinal plant Isatis tinctoria L. were submitted to a broad in vitro screening against 16 anti-inflammatory targets. Dichloromethane (DCM) extracts from dried leaves showed a marked cyclooxygenase (COX) inhibitory activity with a preferential effect on COX-2 catalysed prostaglandin synthesis. A supercritical fluid extraction (SFE) procedure employing CO2-modifier mixtures was developed by which the bioactivity profile and chromatographic fingerprint of the DCM extract could be reproduced. High-resolution activity directed on-line identification of the COX-2 inhibitory principle, using a combination of LC-DAD-MS with a microtitre-based bioassay, led to the identification of tryptanthrin (1) as the constituent responsible for essentially all COX-2 inhibitory activity in the crude extract. Following on-line identification, 1 was isolated at preparative scale and its structure confirmed by comparison with synthetic tryptanthrin. In an assay with lipopolysaccharide stimulated Mono Mac 6 cells, tryptanthrin (1) was of comparable potency (IC50 = 64 nM) than the preferential COX-2 inhibitors nimesulide (IC50 = 39 nM) and NS 398 (IC50 = 2 nM). The SFE extract and 1 showed no cytotoxicity in Mono Mac 6 and RAW 264.7 cells when tested at 100 microg/ml and 10 microM, respectively.

A new 7,8-euphadien-type triterpenoid from Brackenridgea nitida and Bleasdalea bleasdalei that inhibits DNA polymerase beta.

Bioassay-guided fractionation of extracts prepared from Brackenridgea nitida and Bleasdalea bleasdalei, using an assay to detect DNA polymerase beta inhibition, resulted in the isolation of the inhibitory principle, (24E)-3beta-hydroxy-7,24-euphadien-26-oic acid (1), a new euphane triterpenoid. The structure of 1 was established on the basis of HRMS and 1D and 2D NMR spectroscopic methods and was confirmed further by X-ray crystallographic analysis. Compound 1 inhibited rat DNA polymerase beta with an IC(50) value of 23 microM in the presence of bovine serum albumin (BSA) and 9.7 microM in the absence of BSA, consistent with the possibility that 1 may be of utility in vivo. This possibility was further supported by the finding that 1 potentiated the inhibitory action of the anticancer drug bleomycin in cultured P-388D(1) cells, reducing the number of viable cells by 48% when employed at a concentration of 25 microM in the presence of an otherwise nontoxic (75 nM) concentration of bleomycin. Compound 1 is the first euphane-type triterpenoid found to inhibit DNA polymerase beta.

DNA polymerase beta inhibitors from Baeckea gunniana.

Crude plant extracts were surveyed for their ability to inhibit DNA polymerase beta. A methyl ethyl ketone extract prepared from Baeckea gunniana was identified as a potent inhibitor of the enzyme. Bioassay-guided fractionation of the extract, using an assay to monitor the inhibitory potential of individual fractions toward DNA polymerase beta, led to the isolation of four active ursane and oleanane triterpenoids (1-4). Inhibitory principle 1 is a new natural product, and 2 is a novel compound. Their structures were established as 3 beta-hydroxyrus-12,19(29)-dien-28-oic acid (1) and 3 beta-hydroxyrus-18,20(30)-dien-28-oic acid (2) by spectroscopic analysis and by comparison with the data for the structurally related compound ursolic acid (4). Also isolated as a DNA polymerase beta inhibitor was oleanolic acid (3). Compounds 1-4 had IC50 values of 5.3-8.5 microM as inhibitors of polymerase beta in the presence of bovine serum albumin (BSA) and 2.5-4.8 microM in the absence of BSA.

A dual inhibitor of platelet-derived growth factor beta-receptor and Src kinase activity potently interferes with motogenic and mitogenic responses to PDGF in vascular smooth muscle cells. A novel candidate for prevention of vascular remodeling.

PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of Src kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF alpha-receptor was susceptible, albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.

Antifungal Activity of an Aqueous Extract of Leaves of Garlic Creeper (Adenocilymma alliaceum Miers.)

The antifungal activity of an aqueous extract of the leaves of garlic creeper (Adenocalyxmna alliaceum Miers.) was tested against several fungi. The extract showed a broad fungitoxic spectrum as tested by inhibition of spore germination. Exposure of the spores of Alterniaria brassicae (Berk.) Sacc. to the extract for 5 min inhibited the spore germination by 72%, and after exposure for 10 min, a 100% inhibition was found. The inhibitory principle of the extract had a good shelf life at 10°C, and was thermostable when kept for 1 h at 60°C and on boiling at 100°C for 3 min. However, the inhibitory activity of the extract decreased to 70% on boiling for 5 min and was completely lost after boiling for 10 min.

Tissue factor pathway inhibitor: from unknown coagulation inhibitor to major antithrombotic principle.

Time for primary review 20 days. Acute thrombosis is the major immediate cause of cardiovascular death. The thrombotic process is also a stimulus for developing and continuing the atherosclerotic process. To control thrombus formation has been the aim of scientists during several decades. Exposure of tissue factor (TF) to blood, for instance on rupture of atherosclerotic plaques or on endothelial cell damage, may cause acute obstruction of a vessel due to thrombus formation. Thus, the physiological TF inhibitor, tissue factor pathway inhibitor (TFPI), is of interest both pathophysiologically and for its pharmacological potential in inhibiting TF-induced thrombus formation. Exposure of TF to circulating blood starts the coagulation cascade by binding to Factor VII(a) (FVIIa), and the complex of FVIIa and TF activates Factor X (FX) and Factor IX (FIX). TFPI is dependent on some degree of coagulation activation and formation of activated FX (FXa) in order to inhibit the FVIIa/TF complex. Physiologically, TFPI's main role is inhibition of small amounts of tissue factor, and this inhibition is probably essential for maintaining a normal hemostatic balance. It is not known whether total lack of this inhibitor is compatible with life, since no such deficiency has been found. Serial measurements of increasing plasma TFPI during the course of a disease may signal poor prognosis. Heparin accelerates TFPI's inhibitory effect and free TFPI has great heparin affinity. TFPI probably contributes significantly to the anticoagulant properties of the endothelium, and may be particularly important for the outcome of vascular injury. The discovery of the inhibitory principle of TFPI has opened a new arena for pharmacological intervention in the thrombotic process. This may well be a major breakthrough for the control of acute thrombosis in cardiovascular disease. Recombinant TFPI has proved effective in the treatment of experimental disseminated intravascular coagulation, sepsis and thrombosis. Whether TFPI …

Putative mechanism and dynamics of inhibition of the apple scab pathogen Venturia inaequalis by compost extracts

Clarified water extracts of slurries of spent mushroom substrate (SMS) inhibited in vitro germination of conidia of the apple scab pathogen Venturia inaequalis by up to 98% relative to germination in water controls. Inhibition of conidial germination increased with incubation time of slurries over 5 to 7 d and persisted for at least 14 d, at which time experiments were terminated. Compost slurries became anaerobic within 1 h when incubated without aeration. Aeration of slurries decreased efficacy of the resulting extracts compared to non-aerated controls. When aerated slurries were allowed to incubate without further aeration, extracts regained efficacy, becoming not significantly different from non-aerated controls. There was no difference in efficacy between filtered (0.1μm) and untreated extracts. Passage of filtrate through microconcentrators with molecular cut-off limits ranging from 100 to 3 kDa did not diminish activity of the extracts. Autoclaved extracts were less effective than mutreated extracts but retained most of their efficacy. Extracts produced from sterile SMS were virtually ineffective compared with those from non-sterile SMS. When small volumes of slurry from raw SMS were added to slurries of sterile SMS and incubated for an additional period, efficacy of the inoculated SMS was enhanced compared to uninoculated controls. We conclude that a major inhibitory principle of the SMS extract is a low molecular weight, heat-stable, non-protein metabolite produced by anaerobic microorganisms in the compost.

Biliary excretion of biochemically active cyanobacteria (blue-green algae) hepatotoxins in fish

Previous reports demonstrated that microcystin and related cyanobacteria polypeptides are rapidly cleared from plasma and accumulate in liver tissue. In the present study, we have used their ability to inhibit protein phosphatases to show that these cyanobacteria hepatotoxins are excreted into the bile of experimentally poisoned rainbow trout. At various times after oral administration of hepatotoxic Microcystis aeruginosa, bile samples were analysed for microcystin content by methanol extraction and protein phosphatase assays. An inhibitory principle that specifically suppressed protein phosphatase activity was detected in all bile samples removed between 1 and 72 h after oral exposure to toxic algae. These results indicate that biochemically active microcystin molecules are excreted into the biliary tract of poisoned fish.

Antichlamydial activity of vaginal secretion

OBJECTIVE: The purpose of this study was to investigate the possible antichlamydial activity of vaginal secretion and to partially characterize the inhibitory principle. STUDY DESIGN: Vaginal secretions obtained from 156 women attending a family planning or gynecologic outpatient clinic for contraceptive advice were studied for the influence on the inclusion formation of Chlamydia trachomatis in cycloheximide-treated McCoy cell cultures. RESULTS: Vaginal secretions from 156 women inhibited the inclusion formation of Chlamydia trachomatis. The inhibitions was concentration dependent and the inhibitory principle had a molecular weight of <10,000 d. It was heat labile. It was not related to antichlamydial antibodies in vaginal secretions. Only three (2%) of the women had a positive culture for Chlamydia trachomatis. Three had immunoglobulin A and three had immunoglobulin G antichlamydial antibodies in vaginal secretions. Secretions of those with a vaginal pH of 3.5 to 4.5 decreased the chlamydial inclusion count by 75% compared with controls. The corresponding percentage for those with a pH of 5.0 to 6.0 was 48% and for those with pH >6 was 33%. Vaginal secretions of oral contraceptive users and nonusers did not differ in the capacity to decrease the chlamydial inclusion count, p > 0.01. CONCLUSIONS: When vaginal secretions were added to McCoy cell cultures infected by Chlamydia trachomatis, the chlamydia inclusion number decreased. There was a correlation between pH of the vaginal secretion and the inhibitory principle. Oral contraceptive use had no influence on the inhibition.