Analysis of the Structural Gene Encoding a Hemolysin in Vibrio mimicus

An environmental isolate of V. mimicus, strain E-33, has been reported to produce and secrete a hemolysin of 63 kDa. The hemolysin is enterotoxic in test animals. The nucleotide sequence of the structural gene of the hemolysin was determined. We found a 2,232 bp open reading frame, which codes a peptide of 744 amino acids, with a calculated molecular weight of 83,903 Da. The sequence for the structural gene was closely related to the V. cholerae el tor hlyA gene, coding an exocellular hemolysin. The amino terminal amino-acid sequence of the 63 kDa hemolysin, purified from V. mimicus, was determined by the Edman degradation method and found to be NH₂-S-V-S-A-N-N-V-T-N-N-N-E-T. This sequence is identical from S-152 to T-164 predicted from the nucleotide sequence. So, it seems that the mature hemolysin in V. mimicus is processed upon deleting the first 151 amino acids, and the molecular mass is 65,972 Da. Analyzing the deduced amino-acid sequence, we also found a potential signal sequence of 24 amino acids at the amino terminal. Our results suggest that, like V. cholerae hemolysin, two-step processing also exists in V. mimicus hemolysin.     

Purification and characterization of Treponema hyodysenteriae hemolysin

A hemolysin produced by Treponema hyodysenteriae ATCC27164 was purified from broth filtrates by acetic and (NH₄)₂SO₄ precipitations followed by ion exchange chromatography on diethylaminoethyl-Sephacel and gel filtration using Ultrogel AcA44. The purified hemolysin displayed only one band on polyacrylamide gel electrophoresis. By gel filtration the molecular weight was estimated as 74,000 daltons. The isolated hemolysin was oxygen resistant, heat labile and was not inactivated over a wide range of pH values. Further analysis indicated that this hemolysin was probably a polypeptide or a protein associated with lipids and nucleotides. Its action on rabbit erythrocytes which did not require any divalent cations could not be related to a lipolytic or proteolytic activity.     

Extracellular PagC-HlyAS fusion protein for the generation and identification of Salmonella-specific antibodies

The outer membrane protein, PagC, of Salmonella typhimurium was converted into a secreted protein by linking the 61-amino-acid long, C-terminal signal sequence of the E. coli hemolysin protein (HlyA_S) to the mature PagC peptide. This PagC-HlyA_S fusion protein was expressed and efficiently secreted into the culture supernatant by E. coli upon complementation with the hemolysin secretion proteins HlyB and HlyD. Polyclonal antibodies raised against this fusion protein not only recognized PagC in the membrane fraction of all salmonellae by Western blotting, but also reacted with proteins of smaller size in other gram-negative bacteria tested. A monoclonal antibody against the PagC-HlyA_S fusion protein recognized only PagC in membrane fractions. The antibody-binding domain was determined using synthetic peptides derived from specific PagC domains. Sera from Salmonella-infected human patients and from a rabbit infected with S. typhimurium did not react with PagC in immunoblots, suggesting that PagC may not be recognized as a major antigen by the humoral immune system. Received: 16 August 1995/Received revision: 6 November 1995/Accepted: 10 November 1995     

A novel extracellular protease of Vibrio mimicus that mediates maturation of an endogenous hemolysin

Vibrio mimicus, a human pathogen that causes gastroenteritis, produces an enterotoxic hemolysin as a virulence factor. The hemolysin is secreted extracellularly as an inactive protoxin and converted to a mature toxin through removal of the N‐terminal propeptide, which comprises 151 amino acid residues. In this study, a novel protease having the trypsin‐like substrate specificity was purified from the bacterial culture supernatant. The N‐terminal amino acid sequence of the purified protein was identical with putative trypsin VMD27150 of V. mimicus strain VM573. The purified protease was found to cause maturation of the protoxin by cleavage of the Arg¹⁵¹? Ser¹⁵² bond. Deletion of the protease gene resulted in increased amounts of the protoxin in the culture supernatant. In addition, expression of the hemolysin and protease genes was detected during the logarithmic growth phase. These findings indicate that the protease purified may mediate maturation of the hemolysin.

     

Studies on the factors affecting the growth and hemolytic activity of <Emphasis Type="Italic">Anabaena variabilis</Emphasis>

The hemolytic activity of the cell-free culture supernatant of Anabaena variabilis OL S1 was investigated using the hemolysis of rabbit erythrocytes as an assay. The culture medium of A. variabilis started to exhibit hemolytic activity at the late exponential growth phase, and maximized at the stationary phase. The hemolytic toxin is heat-stable and can be extracted in dichloromethane. The hemolytic activities under different temperature, light intensity and pH showed a high correlation with the cell densities (r=0.965, 0.951, 0.865, respectively), and the optimum condition is 28~30°C, pH 7.5~8.0, light intensity 120 μmol photons m⁻²s⁻¹. The addition of 10~20 μg mL⁻¹ chloramphenicol, an inhibitor of protein synthesis, exhibited no marked suppression on the hemolytic activity. The supplement of 1~20 μg mL⁻¹ glycerol increased the hemolytic activity significantly, suggesting that synthesis of hemolysin was dependent on carbohydrate and lipid metabolism. The spectrum of erythrocyte sensitivity to the hemolysin indicated that rabbit erythrocytes were more sensitive to the hemolysin than were rat and human erythrocytes. Goldfish and cat erythrocytes were, however, insensitive to the hemolytic toxin of A. variabilis.     

Genetic relatedness among tdh+ and trh+ Vibrio parahaemolyticus cultured from Gulf of Mexico oysters (Crassostrea virginica) and surrounding water and sediment

Pathogenic Vibrio parahaemolyticus (Vp) (tdh ⁺/trh ⁺) represent a small percentage of environmental Vp populations, and very little is known about this subpopulation. Repetitive extragenic palindromic PCR and multilocus sequence analysis revealed heterogeneity among 41 Vp containing thermostable direct hemolysin (tdh) and tdh-related hemolysin (trh) that were isolated from Mississippi coastal environments from October 2006 to April 2007. There was no source-specific sequestering in oysters, water, or sediment.     

Expression of the hemolysin operon in Escherichia coli is modulated by a nucleoid-protein complex that includes the proteins Hha and H-NS

The Escherichia coli protein Hha is a temperature- and osmolarity-dependent modulator of the expression of the hemolysin operon. The Hha protein was purified and its DNA-binding properties analyzed. Hha binds in a non-specific manner throughout the upstream regulatory region of the hemolysin operon in the recombinant hemolytic plasmid pANN202-312. A search for interacting proteins revealed that Hha interacts with H-NS. DNA-binding studies showed that, in vitro, Hha and H-NS together form a complex with DNA that differs from those formed with either protein alone. These data, together with the effects of hha and hns mutations on the expression of the hemolysin genes, suggest that in vivo H-NS and Hha form a nucleoid-protein complex that accounts for the thermo-osmotic regulation of the hemolysin operon in E. coli. Received. 18 October 1999 / Accepted: 21 December 1999     

Variation in gut microbial contribution of essential amino acids to host protein metabolism in a wild small mammal community

Herbivory is a dominant feeding strategy among animals, yet herbivores are often protein limited. The gut microbiome is hypothesized to help maintain host protein balance by provisioning essential macromolecules, but this has never been tested in wild consumers. Using amino acid carbon (δ¹³C) and nitrogen (δ¹⁵N) isotope analysis, we estimated the proportional contributions of essential amino acids (AA_ESS) synthesized by gut microbes to five co-occurring desert rodents representing herbivorous, omnivorous and insectivorous functional groups. We found that herbivorous rodents occupying lower trophic positions (Dipodomys spp.) routed a substantial proportion (~40%–50%) of their AA_ESS from gut microbes, while higher trophic level omnivores (Peromyscus spp.) and insectivores (Onychomys arenicola) obtained most of their AA_ESS (~58%) from plant-based energy channels but still received ~20% of their AA_ESS from gut microbes. These findings empirically demonstrate that gut microbes play a key functional role in host protein metabolism in wild animals.     

Energy equivalents of oxygen consumption in animal energetics

Summary The energy equivalents given in the literature are reviewed and criticised. New equivalents are calculated for protein respiration.The energy equivalent for converting rate of oxygen consumption into rate of heat production (Q _ox cal mg^-1 oxygen consumed) is 3.53 cal mg^-1 for carbohydrate oxidation, 3.28 cal mg^-1 (range 3.22–3.32) for fat oxidation. Q _ox values for the respiration of standard protein are the same at 3.25 cal mg^-1 for ureotelic and uricotelic animals, and about 2% less at 3.20 cal mg^-1 for ammoniotelic animals. The energy equivalent for converting rate of oxygen consumption into rate of energy loss in excreta (Q _ex cal mg^-1) varies considerably with different excretory products. Values for standard protein are 0.62 cal mg^-1 for ammonioteles, 0.58 cal mg^-1 for ureoteles, and 0.94 cal mg^-1 for uricoteles.The various factors affecting both Q _ox and Q _ex are discussed, and examples of the estimation of general energy equivalents are given.     

Transferrin-receptor interaction and iron uptake by reticulocytes of vertebrate animals — a comparative study

Summary Transferrin-receptor interactions and iron uptake were studied in eleven different species of vertebrate animals (3 eutherian mammals, 3 marsupials, 2 reptiles and 1 bird, amphibian and bony fish). In the initial experiments it was shown that the uptake of transferrin-bound iron by immature erythroid cells from marsupial and reptilian species occurs by receptor-mediated endocytosis as in other vertebrate animals.Reticulocytes were incubated with¹²⁵I-⁵⁹Fe-labelled transferrins from heterologous species and the results for iron and transferrin uptake compared with those obtained with the homologous protein. Cells from eutherian mammals were able to take up transferrin and iron from other eutherians and from the bob-tailed lizard but not from marsupials and other submammalian species. With marsupials and reptiles a similar specificity was observed, and the marsupial cells could also utilize chicken transferrin but not vice versa.The results were extended by performing competition experiments in which the cells were incubated with radiolabelled homologous transferrin in the presence of increasing concentrations of non-radioactive heterologous transferrins. From the ability of the heterologous proteins to inhibit uptake of the homologous protein relative association constants (K _a ¹) for the transferrin-receptor interactions could be calculated. TheseK _a ¹ values reflected the patterns observed in the first series of experiments.These studies demonstrate that, although specificity exists in transferrin-receptor interactions throughout the range of vertebrate animals, in several instances reactivity between widely divergent species is also observed. Hence, structural similarities have been maintained throughout evolution. Nevertheless, no evidence of interaction between transferrin and its receptor from the two divisions of the Mammalia, the eutherians and the marsupials, was observed.Abbreviations BSS Hanks balanced salt solution - PBS phosphate-buffered saline - RRS Rana Ringer solution     

Effects of physical exercise and aging on ascorbic acid and superoxide anion levels in peritoneal macrophages from mice and guinea pigs

The relationship of physical activity and aging, two processes with a high production of oxygen-free radicals to the ascorbate and superoxide anion (O ₂ ^- ) contents of peritoneal macrophages was studied in two animal species: guinea-pig (in which ascorbic acid is a vitamin) and mouse (in which ascorbic acid is not a vitamin). The effects of exhaustive exercise were examined in young and old animals. The results show that macrophages from old animals have a lower ascorbate content than those from young ones, whereas with exercise the ascorbate content increased in both old and young animals. This increase was higher in young than in old animals, and more evident in mice than in guinea-pigs. Aging also resulted in an increase in the O ₂ ^- levels of macrophages. With exercise these levels decreased in young mice but increased in young guinea-pigs. In old animals the exhaustive exercise did not change the O ₂ ^- levels. The results suggest in general a lack of correlation between the intracellular ascorbate and O ₂ ^- levels in relation to both physical exercise and aging.Abbreviations PBS phosphate buffered saline - NBT nitroblue tetrazolium - PEC peritoneal exudate cells - PMN polymorphonuclear     

Isotopic reconstruction of human diet and animal husbandry practices during the Classical-Hellenistic, imperial, and Byzantine periods at Sagalassos, Turkey

An isotopic reconstruction of human dietary patterns and livestock management practices (herding, grazing, foddering, etc.) is presented here from the sites of Düzen Tepe and Sagalassos in southwestern Turkey. Carbon and nitrogen stable isotope ratios were determined from bone collagen extracted from humans (n = 49) and animals (n = 454) from five distinct time periods: Classical‐Hellenistic (400–200 BC), Early to Middle Imperial (25 BC–300 AD), Late Imperial (300–450 AD), Early Byzantine (450–600 AD), and Middle Byzantine (800–1200 AD). The humans had protein sources that were based on C₃ plants and terrestrial animals. During the Classical‐Hellenistic period, all of the domestic animals had δ¹³C and δ¹⁵N signatures that clustered together; evidence that the animals were herded in the same area or kept in enclosures and fed on similar foods. The diachronic analysis of the isotopic trends in the dogs, cattle, pigs, sheep, and goats highlighted subtle but distinct variations in these animals. The δ¹³C values of the dogs and cattle increased (reflecting C₄ plant consumption) during the Imperial and Byzantine periods, but the pigs and the goats displayed little change and a constant C₃ plant‐based diet. The sheep had a variable δ¹³C pattern reflecting periods of greater and lesser consumption of C₄ plants in the diet. In addition, the δ¹⁵N values of the dogs, pigs, cattle, and sheep increase substantially from the Classical‐Hellenistic to the Imperial periods reflecting a possible increase in protein consumption, but the goats showed a decrease. Finally, these isotopic results are discussed in the context of zooarcheological, archeobotanical, and trace element evidence. Am J Phys Anthropol 149:157–171, 2012. © Wiley Periodicals, Inc.     

The erythrocyte receptor for Fusobacterium necrophorum hemolysin: phosphatidylcholine as a possible candidate

An attempt was made to determine the receptor for the hemolysin of Fusobacterium necrophorum using horse erythrocyte or its membranes as target. The spectrum of erythrocyte sensitivity has indicated that horse, dog and mouse erythrocytes are highly sensitive whereas cattle, sheep, goat and chicken red blood cells are insensitive to this hemolysin. A high correlation between sensitivity and phosphatidylcholine content of the erythrocyte membranes was noted. Binding of hemolysin to horse erythrocyte membranes was reduced significantly by prior treatment of membranes with phospholipase A₂ but not with phospholipase C. Pretreatment of erythrocyte membranes with pronase, proteinase K, trypsin or neuraminidase did not alter binding of hemolysin to the membranes, suggesting that protein or sialyl residues are not involved as receptors. Gas liquid chromatography analysis showed that the fatty acid profile from hydrolysis of bovine liver phosphatidylcholine by hemolysin and phospholipase A₂ were similar. In conclusion, this report presents evidence that phosphatidylcholine may be acting as a possible receptor for the hemolysin of F. necrophorum.     

THE EFFECT OF TRIETHYLLEAD ON TOTAL AND MYELIN PROTEIN SYNTHESIS IN RAT FOREBRAIN SLICES

Rats (20-day-old) were acutely intoxicated with triethyllead and their forebrains were studied during the following 14 days. All the lead in the tissue was found in the form of triethyllead, proving that the toxin per se was responsible for the pathological changes observed in the organ. The incorporation of [¹⁴C]leucine into the acid-insoluble protein was suppressed in the forebrain slices prepared from the intoxicated animals as well as in the slices, to which PbEt₃ was added in vitro. In both systems the synthesis of myelin protein was inhibited more than the total protein synthesis. The results suggest a specificity of triethyllead toward processes involved in the furnishing of the myelin membrane proteins.     

Pathogenicity ofAeromonas hydrophila in red leg disease in frogs

Endotoxin and hemolysin fromAeromonas hydrophila A₃ were studied to understand the pathogenicity of the organism. Neither the endotoxin nor the hemolysin alone produced typical red leg disease symptoms or death in frogs, even at a very high dosage of 8,000 μg; however, endotoxin and hemolysin together did. Further, histamine-stressed frogs died from hemolysin but not endotoxin. Hemolytic activity of hemolysin increased in cells that were preincubated with endotoxin. Results point to the conclusion that red leg disease in frogs represents a complex interaction between endotoxin and hemolysin and that stress-producing factors other than the endotoxin might trigger disease production.     

Assessment of the Significance of Virulence Factors of Uropathogenic Escherichia coli in Experimental Urinary Tract Infection in Mice

Four Escherichia coli strains, isolated from cystitis patients, belonging to serotype O2:H^? and possessing different combinations of urovirulence factors were examined in an experimental pyelonephritis mouse model to assess the relative importance of virulence factors in causation of urinary tract infections (UTI). The results suggest not only that the each virulence factor has a role in causation of UTI but also that the presence of P fimbriae and production of hemolysin significantly reduced the LD₅₀ and ID₅₀ of the strains in the mouse model. The results also demonstrate that the presence of additional virulence factors acts in an additive or synergetic fashion enhancing the cumulative impact of the strain.     

Ancient Origin of Four-Domain Voltage-gated Na<Superscript>+</Superscript> Channels Predates the Divergence of Animals and Fungi

The four-domain voltage-gated Na⁺ channels are believed to have arisen in multicellular animals, possibly during the evolution of the nervous system. Recent genomic studies reveal that many ion channels, including Na⁺ channels and Ca²⁺ channels previously thought to be restricted to animals, can be traced back to one of the unicellular ancestors of animals, Monosiga brevicollis. The eukaryotic supergroup Opisthokonta contains animals, fungi, and a diverse group of their unicellular relatives including M. brevicollis. Here, we demonstrate the presence of a putative voltage-gated Na⁺ channel homolog (TtrNa_V) in the apusozoan protist Thecamonas trahens, which belongs to the unicellular sister group to Opisthokonta. TtrNa_V displays a unique selectivity motif distinct from most animal voltage-gated Na⁺ channels. The identification of TtrNa_V suggests that voltage-gated Na⁺ channels might have evolved before the divergence of animals and fungi. Furthermore, our analyses reveal that Na_V channels have been lost independently in the amoeboid holozoan Capsaspora owczarzaki of the animal lineage and in several basal fungi. These findings provide novel insights into the evolution of four-domain voltage-gated ion channels, ion selectivity, and membrane excitability in the Opisthokonta lineage.     

Impact of Ginkgo Biloba Extract EGb 761 on Ischemia/Reperfusion – Induced Oxidative Stress Products Formation in Rat Forebrain

Dysbalance in reactive oxygen/nitrogen species is involved in the pathogenesis of cerebral ischemia/reperfusion injury (IRI). Ginkgo biloba extract (Egb 761) pre-treatment was used to observe potential antioxidant/neuroprotective effect after global ischemia/reperfusion. Egb 761 significantly decreased the level of lipoperoxidation (LPO) in rat forebrain total membrane fraction (homogenate) induced by in vitro oxidative stress (Fe²⁺+H₂O₂). In animals subjected to four-vessel global ischemia for 15 min and 2–24 h reperfusion the EGb pretreatment slightly decreased LPO in forebrain homogenate. However, as detected in EGb treated group, the LPO-induced lysine conjugates are attenuated in comparison to non-treated IRI animals. EGb significantly improved parameters which indicate forebrain protein oxidative damage after IRI. The intensity of tryptophane fluorescence was increased by the 18.2% comparing to non-treated IRI group and bityrosine fluorescence was significantly decreased in ischemic (21%) and 24 h reperfused (15.9%) group in comparison non-treated IRI group. In addition, the level of total free SH- groups in pre-treated animals was significantly higher comparing to non-treated animals. Our results indicate that extract of EGb 761 has potent antioxidant activity and could play a role to attenuate the IRI-induced oxidative protein modification and lipoperoxidation in the neuroprotective process.     

Hemolysin II is more characteristic of Bacillus thuringiensis than Bacillus cereus

To investigate the distribution of the hemolysin II determinant among strains of Bacillus cereus and Bacillus thuringiensis, thirteen strains of B. cereus and fourteen strains of B. thuringiensis strains were tested for hybridization of their chromosomal DNAs with a DNA probe containing the B. cereus hemolysin II gene. In addition, the production of hemolysin II, whose activity is not inhibited by cholesterol, was tested. The presence (absence) of the hybridization response in the microorganism's genome correlated with the presence (absence) of cholesterol-unaffected hemolysin production. Only four out of thirteen B. cereus strains were found to give a positive response in hybridization experiments, whereas thirteen out of fourteen B. thuringiensis strains responded positively. DNAs from ten B. thuringiensis strains contained a 3.5 kb EcoRV fragment, which hybridized with the B. cereus hemolysin II gene probe. The 3.5 kb EcoRV DNA fragment from one of these strains (B. thuringiensis VKM-B1555) was cloned and expressed in Escherichia coli cells. The hemolysin encoded by the cloned DNA fragment was not inhibited by cholesterol and possessed all other properties of B. cereus hemolysin II. The obtained data clearly show limited distribution of hemolysin II among B. cereus strains and demonstrate that hemolysin II is more characteristic of B. thuringiensis than B. cereus.