Antibiotic and synergistic effect of Leu-Lys rich peptide against antibiotic resistant microorganisms isolated from patients with cholelithiasis

Pseudomonas aeruginosa has eventually developed resistance against flomoxef sodium, isepamicin and cefpiramide. Therefore, in this study, the antibacterial activity and synergistic effects of the amphipathic-derived P5-18mer antimicrobial peptide were tested against pathogens associated with cholelithiasis that have developed resistance against commonly used antibiotics. The results were then compared with the activities of the amphipathic-derived peptide, P5-18mer, melittin and common antibiotics. Growth inhibition of planktonic bacteria was tested using the National Committee for Clinical Laboratory Standards (NCCLS). The bactericidal activity of the antimicrobial peptides was measured using time-kill curves. Synergistic effects were evaluated by testing the effects of P5-18mer alone and in combination with flomoxef sodium, isepamicin or cefpiramide at 0.5 × MIC. P5-18mer peptide displayed strong activity against pathogens and flomoxef sodium, isepamicin and cefpiramide-resistant bacteria cell lines obtained from a patient with gallstones; however, it did not exert cytotoxicity against the human keratinocyte HaCat cell line. In addition, the results of time-kill curves indicated that P5-18mer peptide exerted bactericidal activity against four strains of P. aeruginosa. Finally, the use of P5-18mer and antibiotics exerted synergistic effects against cell lines that were resistant to commonly used antibiotics. These results indicate that this class of peptides has a rapid microbicidal effect on flomoxef sodium, isepamicin and cefpiramide-resistant strains of P. aeruginosa. Therefore, these peptides may be used as a lead drug for the treatment of acquired pathogens from patients with cholelithiasis who are affected with antibiotic-resistant bacteria.     

Combined Porin Loss and Extended Spectrum β-Lactamase Production is Associated with an Increasing Imipenem Minimal Inhibitory Concentration in Clinical <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> Strains

For this study, 150 clinical isolates of Klebsiella pneumoniae were collected from one hospital in Beijing, China, and assayed for minimal inhibitory concentration (MIC) of imipenem. To elucidate the mechanisms responsible for imipenem MIC variation among extended-spectrum β-lactamase (ESBL)-positive and -negative K. pneumoniae strains, a variety of β-lactamase genes (bla _TEM, bla _CTX-M, bla _SHV, and bla _OXA) were screened by polymerase chain reaction (PCR). The outer membrane profile and expression of related genes (ompK35 and ompK36) then were analyzed and evaluated, respectively. None of the tested isolates were clinically resistant to imipenem, but the range of MICs among ESBL-positive and -negative strains was significantly different. Deficiency in the expression of outer membrane proteins (OmpK35,36) was observed in some of both ESBL-positive(17.6%) and -negative strains (10.9%), but only the ESBL-positive strains depressed by the expression of ompK35/36 had an increased MIC of imipenem (≥0.5 mg/l). These results confirmed that the combination of SHV-1, CTX-M-3, CTX-M-14, TEM-1, or OXA-11 production and reduced expression of ompK35/36 may not result in clinical resistance to imipenem but does correlate with increasing imipenem MIC.     

The effects of bee (Apis mellifera) venom phospholipase A2 on Trypanosoma brucei brucei and enterobacteria

The potential role of phospholipases in trypanosomiasis was investigated using bee venom phospholipase A2 (bvPLA2) as a model. The effects of bvPLA2 on the survival of Trypanosoma brucei brucei, 2 h and 12 h cultures of Enterobacter cloacae, Escherichia coli, Citrobacter freundii were studied. About 1 mg ml⁻¹ bvPLA2 was trypanocidal after 30 min. Some growth occurred at lower concentrations up to 2 h after treatment but viability decreased up to 8 h. Even very low concentrations of bvPLA2 (10⁻¹² mg ml⁻¹) had some trypanocidal activity. Bee venom PLA2 was bactericidal to 2 h bacterial cultures but bacteriostatic to 12 h ones. Minimum bactericidal concentrations were 10⁻⁵-10⁻⁶ mg ml⁻¹. The results showed that bvPLA2 had significant trypanocidal and antibacterial effects on Gram-negative bacteria. The relationship to events occurring during infection is discussed. Phospholipases may play a role in increased endotoxin levels in trypanosomiasis.     

Construction of Opa-Positive and Opa-Negative Strains of Neisseria meningitidis to Evaluate a Novel Meningococcal Vaccine

Neisseria meningitidis is a major global pathogen causing invasive disease with a mortality of 5–10%. Most disease in developed countries is caused by serogroup B infection, against which there is no universal vaccine. Opacity-associated adhesin (Opa) proteins are major meningococcal outer membrane proteins, which have shown recent promise as a potential novel vaccine. Immunisation of mice with different Opa variants elicited high levels of meningococcal-specific bactericidal antibodies, demonstrating proof in principle for this approach. Opa proteins are critical in meningococcal pathogenesis, mediating bacterial adherence to host cells, and modulating human cellular immunity via interactions with T cells and neutrophils, although there are conflicting data regarding their effects on CD4⁺ T cells. We constructed Opa-positive and Opa-negative meningococcal strains to allow further evaluation of Opa as a vaccine component. All four opa genes from N. meningitidis strain H44/76 were sequentially disrupted to construct all possible combinations of N. meningitidis strains deficient in one, two, three, or all four opa genes. The transformations demonstrated that homologous recombination of exogenous DNA into the meningococcal chromosome can occur with as little as 80 bp, and that minor sequence differences are permissible. Anti-Opa bactericidal antibody responses following immunisation of mice with recombinant Opa were specific to the Opa variant used in immunisation. No immunomodulatory effects were observed when Opa was contained within meningococcal outer membrane vesicles (OMVs), compared to Opa-negative OMVs. These observations support the incorporation of Opa in meningococcal vaccines.     

Antibacterial and anti-biofilm effects of cathelicidin peptides against pathogens isolated from cystic fibrosis patients

Six different cathelicidin-derived peptides were compared to tobramycin for antibacterial and anti-biofilm effects against S. aureus, P. aeruginosa, and S. maltophilia strains isolated from cystic fibrosis patients. Overall, SMAP-29, BMAP-28, and BMAP-27 showed relevant antibacterial activity (MIC₅₀ 4-8 μg/ml), and in some cases higher than tobramycin. In contrast, indolicidin, LL-37, and Bac7(1-35) showed no significant antimicrobial activity (MIC₅₀ > 32 μg/ml). Killing kinetics experiments showed that in contrast to tobramycin the active cathelicidin peptides exert a rapid bactericidal activity regardless of the species tested. All three peptides significantly reduced biofilm formation by S. maltophilia and P. aeruginosa strains at 1/2× MIC, although at a lower extent than tobramycin. In addition, BMAP-28, as well as tobramycin, was also active against S. aureus biofilm formation. Preformed biofilms were significantly affected by bactericidal SMAP-29, BMAP-27 and BMAP-28 concentrations, although at a lesser extent than tobramycin. Overall, our results indicate the potential of some cathelicidin-derived peptides for the development of novel therapeutic agents for cystic fibrosis lung disease.     

Inhibition of a cold-active alkaline phosphatase by imipenem revealed by in silico modeling of metallo-β-lactamase active sites

We demonstrate the inhibition of the native phosphatase activity of a cold active alkaline phosphatase from Vibrio (VAP) (IC₅₀ of 44 ± 4 (n = 4) μM at pH 7.0 after a 30 min preincubation) by a specific β-lactam compound (only by imipenem, and not by ertapenem, meropenem, ampicillin or penicillin G). The homologous scaffold was detected by an in silico analysis that established the spatial and electrostatic congruence of the active site of a Class B2 CphA metallo-β-lactamase from Aeromonas hydrophila to the active site of VAP. The tested β-lactam compounds did not inhibit Escherichia coli or shrimp alkaline phosphatase, which could be ascribed to the lower congruence indicated by CLASP. There was no discernible β-lactamase activity in the tested alkaline phosphatases. This is the first time a scaffold recognizing imipenem in an alkaline phosphatase (VAP) has been demonstrated.     

Antifungal susceptibility of epigallocatechin 3-O-gallate (EGCg) on clinical isolates of pathogenic yeasts

This is the first report to investigate the antifungal susceptibility of 21 clinical isolates of seven Candida species to epigallocatechin 3-O-gallate (EGCg) and to compare with six antifungal agents, amphotericin B (AMPH), fluconazole (FLCZ), flucytosin (5FC), itraconazole (ITCZ), micafungin (MCFG), and miconazole (MCZ), using a method following the National Committee for Clinical Laboratory Standards (NCCLS) M27-A guidelines. Among the tested species, Candida glabrata exhibited the highest susceptibility to EGCg (MIC50, 0.5-1 microg/ml and MIC90, 1-2 microg/ml) compared favorably with FLCZ, although they were slightly less susceptible than to AMPH, 5FC, MCFG, ITCZ, and MCZ. Candida guilliemondii and Candida parapsilosis (MIC50, 1-4 microg/ml and MIC90, 2-16 microg/ml) were also susceptible to EGCg, although they appear to be slightly less susceptible to EGCg than C. glabrata and the other antifungal agents tested. Moreover, the susceptibility of Candida krusei strains (MIC50, 2 microg/ml and MIC90, 4-8 microg/ml) to EGCg was approximately 2- to 8-fold higher than those of 5FC and FLCZ. Our data indicate that EGCg can inhibit clinically pathogenic Candida species, although the concentrations of EGCg for antifungal susceptibility were slightly higher than those of tested antifungal agents on the whole. Based on these results, we suggest that EGCg may be effectively used as a possible agent or adjuvant for antifungal therapy in Candidiasis.     

Production of ferulic acid from lignocellulolytic agricultural biomass by Thermobifida fusca thermostable esterase produced in Yarrowia lipolytica transformant

A gene (axe) encoding the AXE thermostable esterase in Thermobifida fusca NTU22 was cloned into a Yarrowia lipolytica P01g host strain. Recombinant expression resulted in extracellular esterase production at levels as high as 70.94 U/ml in Hinton flask culture broth, approximately 140 times higher than observed in a Pichia pastoris expression system. After 72 h of fermentation by the Y. lipolytica transformant in the fed-batch fermentor, the fermentation broth accumulated 41.11 U/ml esterase activity. Rice bran, wheat bran, bagasse and corncob were used as hydrolysis substrates for the esterase, with corncob giving the best ferulic acid yield. The corncob was incubated with T. fusca xylanase (Tfx) for 12 h and then with the AXE esterase for an additional 12 h. Ferulic acid accumulated to 396 μM in the culture broth, a higher concentration than with esterase alone or with Tfx and esterase together for 24 h.     

Determination of protein mobility in mitochondrial membranes of living cells

Molecular mobility in membranes of intracellular organelles is poorly understood, due to the lack of experimental tools applicable for a great diversity of shapes and sizes such organelles can acquire. Determinations of diffusion within the plasma membrane or cytosol are based mostly on the assumption of an infinite flat space, not valid for curved membranes of smaller organelles. Here we extend the application of FRAP to mitochondria of living cells by application of numerical analysis to data collected from a small region inside a single organelle. The spatiotemporal pattern of light pulses generated by the laser scanning microscope during the measurement is reconstructed in silico and consequently the values of diffusion parameters best suited to the particular organelle are found. The mobility of the outer membrane proteins hFis and Tom7, as well as oxidative phosphorylation complexes COX and F₁F₀ ATPase located in the inner membrane is analyzed in detail. Several alternative models of diffusivity applied to these proteins provide insight into the mechanisms determining the rate of motion in each of the membranes. Tom7 and hFis move along the mitochondrial axis in the outer membrane with similar diffusion coefficients (D = 0.7 μm²/s and 0.6 μm²/s respectively) and equal immobile fraction (7%). The notably slower motion of the inner membrane proteins is best represented by a dual-component model with approximately equal partitioning of the fractions (F₁F₀ ATPase: 0.4 μm²/s and 0.0005 μm²/s; COX: 0.3 μm²/s and 0.007 μm²/s). The mobility patterns specific for the membranes of this organelle are unambiguously distinguishable from those of the plasma membrane or artificial lipid environments: The parameters of mitochondrial proteins indicate a distinct set of factors responsible for their diffusion characteristics.     

Effects of Echinaforce treatment on ex vivo-stimulated blood cells

The herb Echinacea purpurea, also called purple coneflower, is regarded as an immune modulator. This study examined changes in cytokine production in blood samples from 30 volunteers before and during 8-day oral administration with an ethanolic extract of fresh Echinacea purpurea (Echinaforce^®). Daily blood samples were ex vivo stimulated by LPS/SEB or Zymosan and analysed for a series of cytokines and haematological and metabolic parameters. Treatment reduced the proinflammatory mediators TNF-α and IL-1β by up to 24% (p < 0.05) and increased anti-inflammatory IL-10 levels by 13% (p < 0.05) in comparison to baseline. This demonstrated a substantial overall anti-inflammatory effect of Echinaforce^® for the whole group (n = 28). Chemokines MCP-1 and IL-8 were upregulated by 15% in samples from subjects treated with Echinaforce^® (p < 0.05). An analysis of a subgroup of volunteers who showed low pre-treatment levels of the cytokines MCP-1, IL-8, IL-10 or IFN-γ (n = 8) showed significant stimulation of these factors upon Echinaforce^® treatment (30-49% increases; p < 0.05), whereas the levels in subjects with higher pre-treatment levels remained unaffected. We chose the term “adapted immune-modulation” to describe this observation. Volunteers who reported high stress levels (n = 7) and more than 2 colds per year experienced a significant transient increase in IFN-γ upon Echinaforce^® treatment (>50%). Subjects with low cortisol levels (n = 11) showed significant down-regulation of the acute-phase proteins IL1-β, IL-6, IL-12 and TNF-α by Echinaforce^® (range, 13-25%), while subjects with higher cortisol levels showed no such down-regulation. This is the first ex vivo study to demonstrate adapted immune-modulation by an Echinacea preparation. While Echinaforce^® did not affect leukocyte counts, we speculate that the underlying therapeutic mechanism is based on differential multi-level modulation of the responses of the different types of leukocytes. Echinaforce^® thus regulates the production of chemokines and cytokines according to current immune status, such as responsiveness to exogenous stimuli, susceptibility to viral infection and exposure to stress.     

Enhancement of viable Campylobacter detection by chemotactic stimuli

The effects of chemotactic stimuli on motility ability of viable Campylobacter to pass through a 0.45 µm pore size filter in viscous condition were investigated. Reference strains including C. jejuni ATCC 33291, C. coli MUMT 18407, C. lari ATCC 43675, and C. upsaliensis DMST 19055 were used. The initial numbers of artificially-inoculated viable cells per g of chicken meat were approximately 10 to 10⁴. Constituents of mucin plus bile (1:1), varieties of amino acids, and sodium salts were added into a soft-agar-coated membrane filter and incubated at both 37 °C and 42 °C for 24 h. The drop plate method was used to determine numbers of viable Campylobacter at 6, 12, 18, and 24 h. After 6 h, constituents of mucin plus bile at the concentrations of 1, 5, and 10% demonstrated significant increases in numbers of viable cells (p < 0.05). The numbers of the organisms at 42 °C were higher than those at 37 °C. In contrast, no significant difference in cell numbers was observed by adding amino acids or sodium salts. In addition, the role of starvation on chemotactic responses was also studied. Starved cells showed lower chemotactic response than non-starved cells. This method permitted rapid detection of viable thermophilic Campylobacter.     

The Enterobacter aerogenes outer membrane efflux proteins TolC and EefC have different channel properties

The outer membrane proteins TolC and EefC from Enterobacter aerogenes are involved in multidrug resistance as part of two resistance-nodulation-division efflux systems. To gain more understanding in the molecular mechanism underlying drug efflux, we have undertaken an electrophysiological characterization of the channel properties of these two proteins. TolC and EefC were purified in their native trimeric form and then reconstituted in proteoliposomes for patch-clamp experiments and in planar lipid bilayers. Both proteins generated a small single channel conductance of about 80 pS in 0.5 M KCl, indicating a common gated structure. The resultant pores were stable, and no voltage-dependent openings or closures were observed. EefC has a low ionic selectivity (P_K/P_Cl = ∼ 3), whereas TolC is more selective to cations (P_K/P_Cl = ∼ 30). This may provide a possible explanation for the difference in drug selectivity between the AcrAB-TolC and EefABC efflux systems observed in vivo. The pore-forming activity of both TolC and EefC was severely inhibited by divalent cations entering from the extracellular side. Another characteristic of the TolC and EefC channels was the systematic closure induced by acidic pH. These results are discussed in respect to the physiological functions and structural models of TolC and EefC.     

Biochemical characterization and structure–function relationship of two plant NCS2 proteins,the nucleobase transporters NAT3 and NAT12 from Arabidopsis thaliana

Nucleobase ascorbate transporters (NATs), also known as Nucleobase:Cation-Symporter 2 (NCS2) proteins, belong to an evolutionary widespread family of transport proteins with members in nearly all domains of life. We present the biochemical characterization of two NAT proteins, NAT3 and NAT12 from Arabidopsis thaliana after their heterologous expression in Escherichia coli UraA knockout mutants. Both proteins were shown to transport adenine, guanine and uracil with high affinities. The apparent K_M values were determined with 10.12 μM, 4.85 μM and 19.95 μM, respectively for NAT3 and 1.74 μM, 2.44 μM and 29.83 μM, respectively for NAT12. Competition studies with the three substrates suggest hypoxanthine as a further substrate of both transporters. Furthermore, the transport of nucleobases was markedly inhibited by low concentrations of a proton uncoupler indicating that NAT3 and NAT12 act as proton–nucleobase symporters. Transient expression studies of NAT-GFP fusion constructs revealed a localization of both proteins in the plasma membrane. Based on the structural information of the uracil permease UraA from E. coli, a three-dimensional experimentally validated homology model of NAT12 was created. The NAT12 structural model is composed of 14 TM segments and divided into two inverted repeats of TM1–7 and TM8–14. Docking studies and mutational analyses identified residues involved in NAT12 nucleobase binding including Ser-247, Phe-248, Asp-461, Thr-507 and Thr-508. This is the first study to provide insight into the structure–function of plant NAT proteins, which reveals differences from the other members of the NCS2 protein family.     

Inhibition of development, swarming differentiation and virulence factors in Proteus mirabilis by an extract of Lithrea molleoides and its active principle (Z,Z)-5-(trideca-4’,7’-dienyl)-resorcinol

Antibacterial activity of Lithrea molleoides extract against Proteus mirabilis has been previously reported by our group. In the present study, the compound (Z,Z)-5-(trideca-4’,7’-dienyl)-resorcinol (1) was isolated as its responsible active principle. The effects of the compound obtained and of L. molleoides extract on P. mirabilis growth and virulence factors were evaluated.Compound 1 showed MIC and MBC values of 4000 μg/ml. It was found that the extract, at four times the MIC, produced complete killing of the uropathogen at 2 h from the beginning of the experiment, while the alkylresorcinol, at four times the MIC, produced the same effect after 24 h. Hemolysis was adversely affected in treatments with both products at 8 μg/ml, while hemagglutination was not altered. The whole extract induced complete autoaggregation of P. mirabilis at 2000 μg/ml, while compound 1 at the same concentration did not show this property. Swarming motility was delayed in treatments with the extract and with 1 at 1000 and 8 μg/ml, respectively, at 8 h from the beginning of the assay. Complete inhibition of the phenomenon was still observed after 24 h when compound 1 was added at 125 μg/ml.These findings offer the possibility of new classes of antimicrobial medicines to tackle infections caused by P. mirabilis.     

Cyclic antimicrobial R-, W-rich peptides: the role of peptide structure and <Emphasis Type="Italic">E. coli</Emphasis> outer and inner membranes in activity and the mode of action

This study compares the effect of cyclic R-, W-rich peptides with variations in amino acid sequences and sizes from 5 to 12 residues upon Gram negative and Gram positive bacteria as well as outer membrane-deficient and LPS mutant Escherichia coli (E. coli) strains to analyze the structural determinants of peptide activity. Cyclo-RRRWFW (c-WFW) was the most active and E. coli-selective sequence and bactericidal at the minimal inhibitory concentration (MIC). Removal of the outer membrane distinctly reduced peptide activity and the complete smooth LPS was required for maximal activity. c-WFW efficiently permeabilised the outer membrane of E. coli and promoted outer membrane substrate transport. Isothermal titration calorimetric studies with lipid A-, rough-LPS (r-LPS)- and smooth-LPS (s-LPS)-doped POPC liposomes demonstrated the decisive role of O-antigen and outer core polysaccharides for peptide binding and partitioning. Peptide activity against the inner E. coli membrane (IM) was very low. Even at a peptide to lipid ratio of 8/1, c-WFW was not able to permeabilise a phosphatidylglycerol/phosphatidylethanolamine (POPG/POPE) bilayer. Low influx of propidium iodide (PI) into bacteria confirmed a low permeabilising ability of c-WFW against PE-rich membranes at the MIC. Whilst the peptide effect upon eukaryotic cells correlated with the amphipathicity and permeabilisation of neutral phosphatidylcholine bilayers, suggesting a membrane disturbing mode of action, membrane permeabilisation does not seem to be the dominating antimicrobial mechanism of c-WFW. Peptide interactions with the LPS sugar moieties certainly modulate the transport across the outer membrane and are the basis of the E. coli selectivity of this type of peptides.     

S-thanatin in vitro prevents colistin resistance and improves its efficacy in an animal model of Pseudomonas aeruginosa sepsis

An experimental study was performed to evaluate the interaction between s-thanatin and colistin both in vitro and in vivo, using two Pseudomonas aeruginosa strains with different patterns of susceptibilities. We evaluated whether selecting for colistin-resistant P. aeruginosa could be prevented in vitro by combining colistin with s-thanatin. The strains were serially exposed in broth to twofold stepwise increasing concentrations of colistin alone or in combination with a fixed concentration [0.25× minimum inhibitory concentration (MIC)] of s-thanatin. We also performed an in vitro synergy study. For in vivo studies, a mouse model of Pseudomonas sepsis has been used. Main outcome measures were lethality and quantitative blood cultures. Exposure to colistin alone gradually selected for Pseudomonas strains with an increased MIC. In vitro studies, s-thanatin showed a positive interaction with colistin, and was able to prevent its resistance. In vivo studies, s-thanatin combined with colistin exhibited the highest efficacy on all main outcome measurements. These results highlight the potential usefulness of this combination and provide a future therapeutic alternative in severe Pseudomonas infections.     

Cell disruption using a different methodology for proteomics analysis of Trypanosoma cruzi strains

We have developed a cell disruption method to produce a protein extract using Trypanosoma cruzi cells based on a straightforward hypoosmotic lysis protocol. The procedure consists of three steps: incubation of the cells in a hypoosmotic lysis buffer, sonication in a water bath, and centrifugation. The final protein extract was designated TcS12. The stages of cell disruption at different incubation times were monitored by differential interference contrast microscopy. After 30 min of incubation in lysis buffer at 4 °C, the T. cruzi epimastigote forms changed from slender to round-shaped parasites. Nevertheless, cell disruption took place following sonication of the sample for 30 min. The efficiency of the methodology was also validated by flow cytometry, which resulted in 72% of propidium iodide (PI)-labeled cells. To estimate the protein extraction yield and the differential protein expression, the proteomics profile of four T. cruzi strains (CL-Brener, Dm28c, Y, and 4167) were analyzed by liquid chromatography tandem mass spectrometry (LCMS/MS) on a SYNAPT HDMS system using the label-free MS^E approach. ProteinLynx Global Server (version 2.5) with Expression^E analysis identified a total of 1153 proteins and revealed 428 differentially expressed proteins among the strains. Gene ontology analysis showed that not only cytosolic proteins but also nuclear and organellar ones were present in the extract.     

10-Å CryoEM Structure and Molecular Model of the Myriapod (Scutigera) 6 × 6mer Hemocyanin: Understanding a Giant Oxygen Transport Protein

Oxygen transport in Myriapoda is maintained by a unique 6 × 6mer hemocyanin, that is, 36 subunits arranged as six hexamers (1 × 6mers). In the sluggish diplopod Spirostreptus, the 1 × 6mers seem to operate as almost or fully independent allosteric units (h ∼ 1.3; P₅₀ ∼ 5 torr), whereas in the swift centipede Scutigera, they intensively cooperate allosterically (h ∼ 10; P₅₀ ∼ 50 torr). Here, we show the chemomechanical basis of this differential behavior as deduced from hybrid 6 × 6mer structures, obtained by single-particle cryo-electron microscopy of the Scutigera 6 × 6mer (10.0 Å resolution according to the 0.5 criterion) and docking of homology-modeled subunits from Scutigera and two diplopods, Spirostreptus and Polydesmus. The Scutigera 6 × 6mer hemocyanin is a trigonal antiprism assembled from six smaller trigonal antiprisms (1 × 6mers), thereby exhibiting D3 point group symmetry. It can be described as two staggered 3 × 6mers or three oblique 2 × 6mers. Topologically, the 6 × 6mer is subdivided into six subunit zones, thereby exhibiting a mantle (24 subunits) and a core (12 subunits). The six hexamers are linked by 21 bridges, subdivided into five types: two within each 3 × 6mer and three between both 3 × 6mers. The molecular models of the 6 × 6mer reveal intriguing amino acid appositions at these inter-hexamer interfaces. Besides opportunities for salt bridges, we found pairs of carboxylate residues for possible bridging via a Ca²⁺ or Mg²⁺ ion. Moreover, we detected histidine clusters, notably in Scutigera, allowing us to advance hypotheses as to how the hexamers are allosterically coupled in centipede hemocyanin and why they act more independently in diplopod hemocyanin.     

Invertebrate pathogenicity and toxin-producing potential of strains of Bacillus thuringiensis endemic to Antarctica

Several strains of Bacillus thuringiensis were previously isolated from soil in Antarctica and appeared to have physiological adaptations to this cold, nutrient-poor environment. In spite of this they could produce abnormally large, parasporal crystals under laboratory conditions. Here, they have been further characterised for toxin genes and invertebrate pathogenicity. All of the strains were positive in PCR assays for the cry1Aa and cry2 genes. This was confirmed by sequence analysis and the parasporal crystals of all strains contained polypeptides of about 130 kDa. This potential for lepidopteran toxicity was borne out in bioassays of purified δ-endotoxins against larvae of Pieris brassicae: the LD₅₀ values of B2408 (288 μg) were comparable to that of the reference strain, HD-12 (201 μg). There was no activity against the nematode Caenorhabditis elegans in spite of the fact that all strains appeared to possess the cry6 gene. PCR screening for genes encoding other nematode-toxic classes of toxins (Cry5, 4 and 21) was negative. B. thuringiensis has never previously been shown to be toxic to Collembola (springtails) but the purified δ-endotoxins of one of the Antarctic strains showed some activity against Folsomia candida and Seira domestica (224 μg and 238 μg, respectively). It seems unlikely that the level of toxicity demonstrated against springtails would support a pathogenic life-style in nature. All of the strains were positive for genes encoding Bacillus cereus-type enterotoxins. In the absence of higher insects and mammals the ecological value of retaining the toxic capability demonstrated here is uncertain.     

Cryopreservation of sperm in farmed Australian greenlip abalone Haliotis laevigata

This study investigated factors important to the development of the liquid nitrogen (LN) vapor sperm cryopreservation technique in farmed greenlip abalone Haliotis laevigata, including (1) cryoprotectant agent (CPA) toxicity; (2) cooling temperature (height above LN surface); (3) thawing temperature; (4) sperm to egg ratio; and (5) sugar supplementation, using sperm motility, fertilization rate or integrity/potential of sperm components and organelles as quality assessment indicators. Results suggested that among the single CPAs evaluated 6% dimethyl sulfoxide (Me2SO) would be the most suitable for sperm cryopreservation in this species. The highest post-thaw sperm motility was achieved with the sperm that had been exposed to LN vapor for 10 min at 5.2 cm above the LN surface, thawed and recovered in 60 and 18 °C seawater bathes, respectively after at least 2 h storage in LN. The highest fertilization rates were achieved at a sperm to egg ratio of 10,000:1 or 15,000:1. Addition of 1% glucose or 2% sucrose produced significantly higher post-thaw sperm motility than 6% Me2SO alone. Among the three cryoprotectant solutions further trialled, 6% Me2SO + 1% glucose produced the highest fertilization rate of 83.6 ± 3.7%. Evaluation of sperm has shown that the addition of glucose could significantly improve the sperm plasma membrane integrity and mitochondrial membrane potential. These results demonstrated a positive role of glucose in the improvement of sperm cryopreservation in farmed greenlip abalone.