Hemolysis Of Rabbit Erythrocytes Research Articles

The Effect of Photoperiod Duration on Humoral Innate and Humoral Adaptive Immune Responsiveness in Campbell’s Dwarf Hamster

We studied the humoral innate immune responsiveness (HII), i.e. a hemolysis of rabbit erythrocytes by complement proteins, and adaptive humoral (antibody production) immune responsiveness (AHI) to SRBC, morphological and hormonal reproductive characteristics and stress level in male Campbell hamsters kept under long-day (LD; 16D: 8N) and short-day (SD; 8D: 16N) photoperiods. The results indicate lower HII, but not AHI in SD. In SD males we observed body mass, anogenital distance, midventral gland size and level of testosterone in the peripheral blood (but not the level of cortisol) decreased. Comparison of SD non-responders, SD responders and LD individuals demonstrated a statistically significant increase of HII in SD photosensitive hamsters, but not in non-responders compared to LD. There was no link between HII and AHI, which indicates an independent photoperiodic responsiveness of different branches of the immune system in Campbell’s hamster.

The choanoflagellate pore-forming lectin SaroL-1 punches holes in cancer cells by targeting the tumor-related glycosphingolipid Gb3

Choanoflagellates are primitive protozoa used as models for animal evolution. They express a large variety of multi-domain proteins contributing to adhesion and cell communication, thereby providing a rich repertoire of molecules for biotechnology. Adhesion often involves proteins adopting a β-trefoil fold with carbohydrate-binding properties therefore classified as lectins. Sequence database screening with a dedicated method resulted in TrefLec, a database of 44714 β-trefoil candidate lectins across 4497 species. TrefLec was searched for original domain combinations, which led to single out SaroL-1 in the choanoflagellate Salpingoeca rosetta, that contains both β-trefoil and aerolysin-like pore-forming domains. Recombinant SaroL-1 is shown to bind galactose and derivatives, with a stronger affinity for cancer-related α-galactosylated epitopes such as the glycosphingolipid Gb3, when embedded in giant unilamellar vesicles or cell membranes. Crystal structures of complexes with Gb3 trisaccharide and GalNAc provided the basis for building a model of the oligomeric pore. Finally, recognition of the αGal epitope on glycolipids required for hemolysis of rabbit erythrocytes suggests that toxicity on cancer cells is achieved through carbohydrate-dependent pore-formation.

HPLC-DAD profiling, enzyme inhibitory, antihemolytic, and photoprotective activities of Limonium delicatulum leaf extract

Limonium delicatulum is a halophyte that synthesizes high levels of various secondary metabolites in response to abiotic stress and therefore can be a valuable source of different bio-active compounds. The leaf extract of L. delicatulum was analyzed for its phenolic compounds by HPLC-DAD and evaluated for its inhibitory effects on acetylcholinesterase (AChE), butyrylcholinesterase (BChE), α-glucosidase, α-amylase, urease, and lipase. The protective effect on oxidative hemolysis of rabbit erythrocytes as well as its photoprotective action have also been investigated. HPLC analysis led to the identification of five phenolic compounds including salvianolic acid B (14.8 mg/g of extract) followed by polydatin (extract 4.0 mg/g of extract). The extract showed strong inhibitory actions on AChE, BChE, α-glucosidase, α-amylase and urease close to or even better than those shown by the standards with IC50 values of 5.94 ± 0.54, 11.68 ± 0.35, 30.17 ± 0.65, 22.74 ± 0.54, and 10.23 ± 087 μg/mL, respectively, while it was inactive against lipase. In addition, an important reduction in the rate of lysis of erythrocytes was observed (with a percentage of 89.18 ± 0.95%) after treatment with leaf extract at the concentration of 200 μg/mL. Finally, an SPF value of 35.5 ± 0.90 was recorded with the leaf extract. These results confirm that L. delicatulum can be of great importance in food, pharmaceutical, and cosmetics.

The development of three ruthenium-based antimicrobial metallodrugs: Design, synthesis, and activity evaluation against Staphylococcus aureus

The development of new classes of antimicrobial is urgently needed due to the widespread occurrence of multi-resistant pathogens. In this study, three novel ruthenium complexes: [Ru(dmob)2(BTPIP)](PF6)2 (Ru(II)-1), [Ru(dbp)2(BTPIP)](PF6)2 (Ru(II)-2), and [Ru(dpa)2(BTPIP)](PF6)2 (Ru(II)-3) (dpa = 2,2’-dipyridylamine, dmob = 4,4’-dimethoxy-2,2’-bipyridyl, dbp = 4,4’-di- tert-butyl-2,2’-dipyridyl, BTPIP = 4-(benzo[ b]thiophen-2-yl)phenyl-1 H-imidazo[4,5- f][1,10]phenanthroline) are synthesized and investigated as antimicrobial metallodrugs. We demonstrate that all three complexes have significant antimicrobial activity against Staphylococcus aureus by testing their minimal inhibitory concentrations = 0.0015–0.0125 mg/mL. The antibacterial activity of the best active complex Ru(II)-3 is 13 times that of ofloxacin (minimal inhibitory concentration = 19.5 μg/mL). Importantly, Ru(II)-3 not only increases the susceptibility of Staphylococcus aureus to existing common antibiotics but also shows noticeably delayed and decreased resistance in Staphylococcus aureus since the minimal inhibitory concentration values of Ru(II)-3 only increased eightfold times after 20 passages. Furthermore, the biofilms formation and rabbit erythrocyte hemolysis assays verified that Ru(II)-3 also efficiently inhibit the biofilm formation and toxin secretion of Staphylococcus aureus.

14-O-[(4,6-Diamino-pyrimidine-2-yl) thioacetyl] mutilin inhibits α-hemolysin and protects Raw264.7 cells from injury induced by methicillin-resistant S. aureus

A new pleuromutilin derivative, 14-O-[(4,6-Diaminopyrimidine-2-yl) thioacetyl] mutilin (DPTM), has been synthesized and proven to be a potent agent against Gram-positive pathogens, especially for Staphylococcus aureus (S. aureus). However, its pharmacological activities against α-hemolysin (Hla), a major virulence factor produced by S. aureus, and inflammations related to S. aureus are still unknown. In the present study, we investigated the DPTM inhibition activities against methicillin-resistant S. aureus (MRSA) Hla and protective efficacy of Raw264.7 cells from injury induced by MRSA. The results showed that DPTM with sub-inhibitory concentrations significantly inhibited Hla on the hemolysis of rabbit erythrocytes and down-regulated the gene expressions of Hla and agrA with a dose-dependent fashion. In Raw264.7 cells infected with MRSA, DPTM efficiently attenuated the productions of lactate dehydrogenase (LDH), nitric oxide (NO) and pro-inflammatory cytokines, as well as the express levels of nuclear factor-kappaB (NF-κB), nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Furthermore, DPTM inhibited the translocation of p-65 to nucleus in RAW264.7 cells infected by MRSA.

Preclinical Characterization of BCX9930, a Potent Oral Complement Factor D Inhibitor, Targeting Alternative Pathway-Mediated Diseases Including Paroxysmal Nocturnal Hemoglobinuria (PNH)

INTRODUCTION: Complement factor D is a serine protease that specifically catalyzes the cleavage of factor B [bound to C3(H2O) or C3b], its only natural substrate, to Ba and Bb. Factor D has the lowest concentration in plasma among all the complement proteins and is the rate-limiting enzyme of the alternative pathway (AP) of complement. Factor D plays a key role in both AP-initiated C3 convertase formation and the amplification loop of the complement cascade. Therefore, targeting factor D is a promising therapeutic strategy to inhibit AP activation for treatment of AP-mediated diseases such as PNH, atypical hemolytic uremic syndrome, and C3 glomerulopathy. Inhibition of terminal complement C5 by eculizumab or ravulizumab results in sustained inhibition of intravascular hemolysis in patients with PNH by blocking formation of the membrane attack complex (C5b-9). However, many PNH patients treated with eculizumab continue to have symptomatic anemia, with up to half of the patients requiring blood transfusions due to extravascular hemolysis as a result of ongoing AP activation and C3 opsonization on erythrocytes. BioCryst Pharmaceuticals has developed a small-molecule, orally bioavailable human factor D inhibitor that is currently under evaluation as oral monotherapy for the treatment of patients with PNH in a Phase 1 study (BCX9930-101; NCT04330534). Here we demonstrate that BCX9930 is a potent and highly selective factor D inhibitor, and completely blocked AP activation on PNH erythrocytes in vitro. METHODS: Factor D enzymatic activity was analyzed in vitro in an esterolytic assay with a synthetic substrate and in a proteolytic assay using its natural substrate factor B bound to C3b. The selectivity was assessed in substrate-specific assays using other human serine proteases. The inhibition of AP-mediated hemolysis was assessed in vitro with rabbit erythrocytes incubated with 10% normal human serum (NHS). The Ham test was used to evaluate the inhibition by BCX9930 of AP-mediated hemolysis of erythrocytes from PNH patients with 20% acidified NHS. Inhibition of AP-mediated C3 fragment deposition was evaluated by flow cytometry using PNH erythrocytes incubated with acidified C5-depleted NHS. Activity of the classical pathway (CP) of complement l was evaluated by measuring 50% Hemolytic Complement (CH50) activity of serum and by assay of CP-mediated hemolysis of antibody-sensitized sheep erythrocytes. Pharmacodynamic effects in rhesus monkeys dosed orally with BCX9930 (100 and 200 mg twice-a-day) were assessed ex vivo with the AP Wieslab assay for the determination of C5b-9 generation after AP activation. RESULTS: BCX9930 potently inhibited esterolytic activity of purified human factor D with a mean 50% inhibitory concentration (IC50) of 14.3 nM. BCX9930 also inhibited its proteolytic activity against factor B bound to C3b with a mean IC50 of 28.1 nM. BCX9930 inhibited AP-mediated hemolysis of rabbit erythrocytes with a mean IC50 of 29.5 nM. Using the Ham test with erythrocytes from three PNH patients, BCX9930 completely suppressed AP-mediated hemolysis with a mean IC50 of 35.4 nM. Furthermore, BCX9930 suppressed C3 fragment deposition on PNH erythrocytes with a mean IC50 value of 39.3nM. BCX9930 did not inhibit enzyme activity of the human serine proteases thrombin, activated protein C, tissue plasminogen activator and trypsin up to 28 μM, and activated factor X and activated factor XII up to 50 μM. The mean IC50 values of BCX9930 inhibition of C1s and plasmin were 936 and 2850 nM, respectively. BCX9930 at a concentration up to 3 μM had no effect on CP activity in in vitro assays. Oral dosing of BCX9930 in monkeys completely suppressed AP activity of serum measured by AP-dependent generation of C5b-9. CONCLUSIONS: BCX9930 inhibitory activity on Factor D in vitro was potent and highly specific. BCX9930 completely blocked hemolysis of PNH cells in vitro and suppressed the accumulation of C3 fragments on PNH erythrocytes, indicating that BCX9930 has the potential to inhibit both intravascular and extravascular hemolysis. After oral dosing of BCX9930 in rhesus monkeys, AP activity was completely suppressed in ex vivo assays. These data demonstrate that BCX9930, an orally administered investigational drug, is a potent and selective factor D inhibitor, and that targeting factor D with BCX9930 has potential for the treatment of patients with PNH and other alternative pathway mediated diseases. Disclosures Chen: BioCryst Pharmaceuticals, Inc.: Current Employment. Cheng:BioCryst Pharmaceuticals, Inc.: Current Employment. Liu:BioCryst Pharmaceuticals, Inc.: Current Employment. Muppa:BioCryst Pharmaceuticals, Inc.: Current Employment. Parker:BioCryst Pharmaceuticals, Inc.: Current Employment. Zhu:BioCryst Pharmaceuticals, Inc.: Current Employment. Raman:BioCryst Pharmaceuticals, Inc.: Current Employment. Kotian:BioCryst Pharmaceuticals, Inc.: Current Employment. Babu:BioCryst Pharmaceuticals, Inc.: Current Employment.

Effects of Antibiotics on α-Toxin Levels during Staphylococcus aureus Culture: Implications for the Protection of Chondrocytes in a Model of Septic Arthritis.

Septic arthritis results from joint infection by Staphylococcus aureus, which produces potent α-toxin causing cell death, potentially leading to permanent cartilage damage. Treatment is by joint irrigation and antibiotics, although it is unclear if, following treatment with antibiotics which cause bacterial lysis, there is release of additional stored α-toxin. A rabbit erythrocyte hemolysis assay was optimised to assess biologically-active α-toxin from cultured S. aureus α-toxin strain DU5946. Hemoglobin release was measured spectrophotometrically following addition of a bacteriostatic antibiotic (linezolid) or a bacteriolytic antibiotic (penicillin). A bovine cartilage model of septic arthritis was used to test the protective effects of antibiotics against S. aureus infection. During S. aureus culture, α-toxin levels increased rapidly but the rate of rise was quickly (within 20 minutes) suppressed by linezolid (25 μg/mL). Penicillin also reduced the increase in α-toxin levels; however, the time course was relatively slow compared to linezolid even at high concentrations (50,000 U/mL). The efficacy of penicillin (250,000 U/mL) at reducing the rise in α-toxin was approximately 8% less than that of linezolid (P < 0.05) suggesting the presence of additional toxin. This could be due to a delayed action of penicillin, and/or release of a small pool of stored α-toxin from dying bacteria. In a bovine cartilage model, however, there was no difference between the protection of in situ chondrocytes against S. aureus by penicillin or linezolid (P > 0.05). The results suggested that equally effective protection of chondrocytes against S. aureus septic arthritis may be obtained by the bacteriostatic or bacteriolytic antibiotics tested.

Optimizing the Composition of Irrigation Fluid to Reduce the Potency of Staphylococcus aureus α-Toxin: Potential Role in the Treatment of Septic Arthritis.

Septic arthritis is commonly caused by Staphylococcus aureus and is a medical emergency requiring antibiotics and joint irrigation. The bacteria produce α-toxin causing rapid cartilage cell (chondrocyte) death. Saline (0.9%NaCl) lavage is normally used to remove bacteria and toxins, however, its composition might be suboptimal to suppress the lethal effects of α-toxin. We utilized rabbit erythrocyte hemolysis as a sensitive, biologically relevant assay of α-toxin levels to determine if changes to osmolarity, temperature, pH, and divalent cation (Mg2+, Ca2+) concentration were protective. Erythrocytes were incubated in the various conditions and then exposed to α-toxin ("chronic" challenge) or incubated with α-toxin and then exposed to experimental conditions ("acute" challenge). Raising osmolarity from 300 mOsm (0.9%NaCl) to 400, 600, or 900 mOsm (sucrose addition) when applied chronically, significantly reduced hemolysis linearly. As an acute challenge, osmotic protection was significant and similar over 400 to 900 mOsm. Reducing temperature chronically from 37°C to 25°C and 4°C significantly reduced hemolysis, however, when applied as an acute challenge although significant, was less marked. Divalent cations (Mg2+, Ca2+ at 5mM) reduced hemolysis. Varying pH (6.5, 7.2, 8.0) applied chronically marginally reduced hemolysis. The optimized saline (0.9% NaCl; 900 mOsm with sucrose, 5 mM MgCl2 (37°C)) rapidly and significantly reduced hemolysis compared with saline and Hank's buffered saline solution applied either chronically or acutely. These results on the effect of S. aureus α-toxin on erythrocytes showed that optimizing saline could markedly reduce the potency of S. aureus α-toxin. Such modifications to saline could be of benefit during joint irrigation for septic arthritis.

Subinhibitory concentrations of resveratrol reduce alpha-hemolysin production in Staphylococcus aureus isolates by downregulating saeRS

Resveratrol is a natural phytoalexin. In recent studies, it has been shown to have beneficial effects on cardiovascular disease and cancer and has been deemed to have effective antiviral and immunomodulatory activities. Methicillin-resistant Staphylococcus aureus is a multidrug-resistant pathogen associated with skin and soft tissue infections. Alpha-hemolysin is known to play a key role in the symptoms caused by S. aureus, and the saeRS two-component system has been shown to be a major regulatory system of S. aureus virulence. The present study was designed to determine the effect of subinhibitory concentrations of resveratrol on the production of alpha-hemolysin in S. aureus. The effect of resveratrol on the transcription of S. aureus was studied by transcriptome sequencing. A total of 760 genes with >2-fold changes in expression were selected, including 479 upregulated genes and 281 downregulated genes. On the basis of transcriptome sequencing, the expression of alpha-hemolysin in the S. aureus strains of the resveratrol-treated group was downregulated. Our results showed that resveratrol weakly inhibited the growth of S. aureus strains, and subinhibitory concentration of resveratrol decreased the expression of hla and inhibited the regulation of saeRS. Hemolysis testing confirmed that resveratrol had an inhibitory effect on the hemolysis of rabbit erythrocytes infected with S. aureus strains in a dose-dependent manner. Resveratrol also decreased the hemolytic capacity by reducing the production of alpha-hemolysin. We found that resveratrol could decrease the expression of hla and reduce the secretion of alpha-hemolysin by downregulating saeRS. These findings have provided more evidence of the potential of resveratrol as a drug for resisting S. aureus infections.

Effects of chloride channels on hemolysis induced by puerarin injection

To observe the effects of blocking and activating chloride channels on hemolysis induced by puerarin injection in rabbits and to investigate the roles of chloride channels in hemolytic reaction induced by puerarin injection. Rabbit erythrocyte suspension was incubated with different concentrations of puerarin injection(0.75, 1.5, 3, 6, 12 mg/ml) at 37oC for 6 hours. The cell imaging system was employed to observe whether puerarin injection induced hemolysis. The hemolysis rate was detected by microplate reader and flow cytometry. Effects of activating and closing chloride channels on the hemolysis induced by puerarin injection were explored. Puerarin injection could induce the hemolysis of rabbit erythrocytes in vitro. In the range of 1.5 mg/ml~12 mg/ml, puerarin injection could induce hemolysis in a concentration-dependent manner (n=3, P<0.01). The chloride channel blockers tamoxifen (20 μmol/L) and ATP (10 mmol/L) significantly inhibited the hemolysis induced by puerarin injection (n=3~5, P<0.01). Application of low concentration ATP (50 μmol/L) to activate the chloride channel significantly increased puerarin injection induced hemolysis (n=4, P<0.01). The hemolytic effect of puerarin injection is dose-dependent in vitro, and the activation of chloride channel is closely related to the hemolysis induced by puerarin injection.

An Ointment Consisting of the Phage Lysin LysGH15 and Apigenin for Decolonization of Methicillin-Resistant Staphylococcus aureus from Skin Wounds.

Staphylococcus aureus (S. aureus) is a common and dangerous pathogen that causes various infectious diseases. Skin damage, such as burn wounds, are at high risk of Staphylococcus aureus colonization and infection, which increases morbidity and mortality. The phage lysin LysGH15 exhibits highly efficient lytic activity against methicillin-resistant S. aureus (MRSA) and methicillin-susceptible S. aureus (MSSA) strains. Apigenin (api) significantly decreases haemolysis of rabbit erythrocytes caused by S. aureus and shows anti-inflammatory function. LysGH15 and api were added to Aquaphor to form an LysGH15-api-Aquaphor (LAA) ointment. The LAA ointment simultaneously exhibited bactericidal activity against S. aureus and inhibited haemolysis. In an LAA-treated mouse model of an MRSA-infected skin wound, the mean bacterial colony count decreased to approximately 102 CFU/mg at 18 h after treatment (and the bacteria became undetectable at 96 h), whereas the mean count in untreated mice was approximately 105 CFU/mg of tissue. The LAA ointment also reduced the levels of pro-inflammatory cytokines (TNF-α, IL-1β, and IFN-γ) and accelerated wound healing in the mouse model. These data demonstrate the potential efficacy of a combination of LysGH15 and api for use as a topical antimicrobial agent against S. aureus.

English

Mussel contains a variety of adhesion proteins, among which, Mytilus galloprovincialis foot protein type 5 (Mgfp-5) is one of the major proteins required for substrate adhesion. The labor-intensive nature and insufficiency of the extraction process have frequently resulted in very little purified recombinant Mgfp-5. These prompt technologies such as chemical synthesis and genetic engineering are employed to overcome these limitations. In this study, successful expression and purification of the recombinant Mgfp-5 using Escherichia coli BL21 (DE3) and affinity chromatography were reported. Production yield of 12.25% and purity of 96.92%, respectively were observed. The 3,4-dihydroxyphenylalanine (DOPA) content (9.60 pmol/g) and the adhesion (1 116 nN) in modified recombinant Mgfp-5 were 9.32 times and 1.6 times as great as those in the unmodified recombinant Mgfp-5, respectively. Recombinant Mgfp-5 at a concentration of 9.6 mg/L had little cytotoxicity on mouse L-929 fibroblast cells, which was toxic at first in cytotoxicity test, and a concentration of not more than 20 μg/mL would not lead to hemolysis of rabbit erythrocytes. In this case, recombinant Mgfp-5 is biosecure, providing the foundation for Mgfp-5 manufacturing as well as the development of clinical biological adhesive. Key words: Mgfp-5, 3,4-dihydroxyphenylalanine (DOPA), adhesion, cytotoxicity, hemolysis.

Combinatorial Synthesis and in Vitro Evaluation of a Biaryl Hydroxyketone Library as Antivirulence Agents against MRSA

Antibiotic resistance coupled with decreased development of new antibiotics necessitates the search for novel antibacterial agents. Antivirulence agents offer an alternative to conventional antibiotics. In this work, we report on a family of small-molecule antivirulence agents against methicillin-resistant Staphylococcus aureus (MRSA), the most widespread bacterial pathogen. Structure-activity relationship studies led to the development of a concise synthesis of a 148-member biarylhydroxyketone library. An acylation bond-forming process afforded resorcinols (1) and aryloxy acetonitriles (2) as synthons. A Lewis-acid-activated Friedel-Crafts' acylation step involving a nitrile functionality of 2 by ZnCl2, followed by nucleophilic attack by 1 was executed to obtain biaryl hydroxyketones in excellent yields. A large number of products crystallized. This strategy affords a range of biarylhydroxyketones in a single step. This is the first collective synthetic study documenting access to this class of compounds through a single synthetic operation. In vitro efficacy of compounds in this library was evaluated by a rabbit erythrocyte hemolysis assay. The most efficacious compound, 4f-12, inhibits hemolysis by 98.1 ± 0.1% compared to control in the absence of the compound.

Antivirulence agents against bacterial pathogens

Resistance to antibiotics has created a severe public health problem worldwide. The development of novel antibacterial agents represents an unmet medical need. An alternative approach to combat bacterial infections is the use of antivirulence agents. These compounds do not kill bacteria as antibiotics do but inhibit the production of toxins and other virulence factors. It is the toxins that cause disease. Prevention of toxin secretion would render the bacteria harmless. We have discovered small-molecule compounds that inhibit the biosynthesis of a series of virulence factors in MRSA strain USA 300, including alpha-toxin, phenol-soluble modulin alpha and RNAIII. These compounds bind to the response regulator AgrA, thereby inhibiting the expression of virulence factors under control of the agr operon. These compounds also inhibit hemolysis of rabbit erythrocytes. One of these compounds is diflunisal, an FDA-approved non-steroidal anti-inflammatory drug. This represents a new use for an old drug. A combination of diflunisal and vancomycin is more efficacious in improving the survival rate in an MRSA animal model of Galleria melonella. In parallel we have discovered antivirulence compounds against Acinetobacter baumannii by targeting the enzyme acyl homoserine lactone synthase that catalyzes the formation of the signaling molecules in Gram-negative bacteria. These compounds inhibit the growth of A. baumannii in 30% horse serum. Antivirulence compounds may be used in the future for prophylaxis against skin infections or as adjuvants in conventional antibiotic therapy.

Cytolytic Peptide Fragments of Cyt1Aa from Bacillus thuringiensis subsp. israelensis

Cyt1Aa is the major and most active component of the parasporal crystal of the Gram-positive soil entomopathogenic bacterium Bacillus thuringiensis subsp. israelensis. The Cyt1Aa protoxin exhibits some hemolytic and cytolytic activity. However, highly active 22-25 kDa toxins are obtained after proteolysis of Cyt1Aa from both the N- and the C-termini. As shown in this study, preliminary binding of the protoxin to polylamellary liposomes or partial denaturation of Cyt1Aa and further processing by several exogenous proteases yielded short 4.9-11.5 kDa cytolytic peptide fragments of Cyt1Aa. The shortest 51 amino acid peptide was obtained after pre-incubation of Cyt1Aa with SDS and proteolysis with proteinase K. This peptide was purified, identified as the Ile87-Asp137 fragment of Cyt1Aa and was shown to exhibit more than 30 % hemolysis of rabbit erythrocytes.

Antioxidant Properties of Indigenous Raw and Fermented Salad Plants

This study evaluates the antioxidant potential of selected plant species by assessing three parameters: antioxidant inhibition of hydrogen peroxide induced hemolysis, free radical scavenging activity and presence of superoxide dismutase activity. Protection against hemolysis was assessed by the rabbit erythrocyte hemolysis assay while free radical scavenging activity was estimated spectophotometrically using 2,2–diphenyl-1-picrylhydrazil as the scavenger. Superoxide dismutase was assessed by measuring the inhibition by superoxide dismutase on the reaction of xanthine oxide generated superoxide radical with a tetrazolium salt. The best protection against hydrogen peroxide induced hemolysis was by extracts of lemon grass (Cymbopogon citratus) leaves. Hempedu bumi (Andrographis paniculata), garlic (Allium sativum) and turmeric (Curcuma domestica) also showed significant anti-hemolytic effect. Good free radical scavenging activity was shown by raw and fermented bean (Phaseolus vulgaris), hempedu bumi (Andrographis paniculata) and ulam raja (Cosmos caudatus). Ulam raja (Cosmos caudatus), lemon grass, hempedu bumi and turmeric showed significant superoxide dismutase activity. The antioxidant potential of Andrographis paniculata was good as evident by the three parameters assayed. However, neem (Azadirachta indica) has limited antioxidant potential. Plants with good antioxidant capabilities include lemon grass and garlic.

Mortalin inhibitors sensitize K562 leukemia cells to complement‐dependent cytotoxicity

Mortalin, the mitochondrial hsp70, is a vital constitutively expressed heat shock protein. Its elevated expression has been correlated with malignant transformation and poor cancer prognosis. Cancer cells exhibit increased resistance to complement-dependent cytotoxicity, partly due to their capacity to eliminate the complement membrane attack complex (MAC) from their cell surface. As we have previously reported, mortalin and the complement membrane attack complexes are released in membrane vesicles from complement attacked cells. As shown here, knock down of mortalin with specific siRNA reduces MAC elimination and enhances cell sensitivity to MAC-induced cell death. Similar results were obtained with MKT-077, a cationic rhodacyanine dye that inhibits mortalin. Treatment of human erythroleukemia K562 and colorectal carcinoma HCT116 cells with MKT-077 sensitizes them to cell death mediated by MAC but not by streptolysin O. Pre-treatment of cells with MKT-077 also reduces the extent of MAC-mortalin vesiculation following a sublytic complement attack. In the presence of MKT-077, the direct binding of mortalin to complement C9, the major MAC component, is inhibited. The tumor suppressor protein p53 is a known mortalin client protein. The effect of MKT-077 on complement-mediated lysis of HCT116 p53(+/+) and p53(-/-) cells was found to be independent on the presence of p53. Our results also demonstrate that recombinant human mortain inhibits complement-mediated hemolysis of rabbit erythrocytes as well as zinc-induced C9 polymerization. We conclude that mortalin supports cancer cell resistance to complement-dependent cytotoxicity and propose consideration of mortalin as a novel target for cancer adjuvant immunotherapy.

A novel mutation in the complement regulator clusterin in recurrent hemolytic uremic syndrome

A novel heterozygous mutation in the clusterin gene, nucleotide position A1298C (glutamine > proline Q433P), was detected in exon 7 of a child with recurrent hemolytic uremic syndrome (HUS). The same mutation was found in the child's two siblings and mother but not in 120 controls. In addition, a previously described heterozygous mutation was detected in the gene encoding membrane cofactor protein (MCP) causing a 6 base-pair deletion 811-816delGACAGT in exon 6. It was found in the patient, both siblings and the father. One sibling had recovered from post-streptoccocal glomerulonephritis. Clusterin levels in the patient, siblings and parents were normal as was the migration pattern in a gel. Patient serum induced C3 and C9 deposition on normal washed platelets, and platelet activation, as detected by flow cytometry. The same phenomenon was found in serum taken from the siblings and the mother but not in the sample from the father and controls. Addition of clusterin to patient serum did not inhibit complement activation on platelets. The Q433P mutant, in isolated form, was further studied by binding to the components of the terminal complement complex. The mutant did not bind to C5b-7 that was immobilized onto a BIAcore chip, whereas wild-type clusterin did, indicating that the mutation could lead to defective inhibition of formation of the membrane attack complex under these conditions. Hemolysis of rabbit erythrocytes was inhibited by wild-type clusterin but not by the mutant. Mutated clusterin could thus not prevent assembly of the membrane attack complex on platelets and erythrocytes.

Design and characterization of novel hybrid peptides from LFB15(W4,10), HP(2-20), and cecropin A based on structure parameters by computer-aided method

The increasing problem of antibiotic resistance among pathogenic bacteria requires development of new antimicrobial agents. The pivotal assets of the antimicrobial peptide include potential for rapid bactericidal activity and low propensity for resistance. The four new antimicrobial hybrid peptides were designed based on peptides LFB15(W4,10), HP(2-20), and cecropin A according to the structure-activity relationship of the amphipathic and cationic antimicrobial peptides. Their structural parameters were accessed by bioinformatics tools, and then two hybrids with the most potential candidates were synthesized. The hybrid peptide LH28 caused an increase in antibiotic activity (MIC(50)=1.56-3.13 microM) against given bacterial strains and did not cause obvious hemolysis of rabbit erythrocytes at concentration of 3.13 microM with effective antimicrobial activity. The results demonstrate that evaluating the structural parameters could be useful for designing novel antimicrobial peptides.

Chemical Inhibition of Alpha-Toxin, a Key Corneal Virulence Factor of Staphylococcus aureus

alpha-Toxin mediates extreme corneal damage during Staphylococcus aureus keratitis. Chemical inhibition of this toxin was sought to provide relief from toxin-mediated pathology. Inhibition of alpha-toxin by phosphate-buffered saline (PBS), 0.1% methyl-beta-cyclodextrin (CD), or CD plus cholesterol (0.1%, CD-cholesterol) was assayed by hemolysis of rabbit erythrocytes. Pathologic changes in rabbit corneas injected with 12 hemolytic units of alpha-toxin suspended in PBS, 1% CD, or 1% CD-cholesterol were compared over time. Rabbit corneas injected with 10(2) colony forming units (CFU) of S. aureus were treated from 7 to 13 hours postinfection (PI) with a total of 15 drops of CD-cholesterol, CD, or PBS. Slit lamp examination (SLE) and measurement of erosions were performed at 13 hours PI and bacteria were quantified at 14 hours PI. Toxin-mediated lysis of erythrocytes was inhibited up to 16,000-fold in the presence of CD-cholesterol compared with CD or PBS. Eyes injected with alpha-toxin mixed with CD-cholesterol had, at 7 hours postinjection, significantly smaller erosions than eyes injected with alpha-toxin in PBS or alpha-toxin mixed with CD (P = 0.0090 and P = 0.0035, respectively). Eyes infected with S. aureus and treated with CD-cholesterol had significantly lower SLE scores than eyes treated with CD or PBS (P <or= 0.0103 and P <or= 0.0017, respectively); however, there were no differences in the number of bacteria present (P >or= 0.0648). CD-cholesterol is a potent inhibitor of alpha-toxin activity in vitro and an effective means to arrest corneal damage during S. aureus keratitis.