Ezrin protects bovine spermatozoa from spontaneous acrosome reaction.

To interact and penetrate the egg, the spermatozoon must undergo a maturation step called the acrosome reaction (AR) in close proximity to the egg. This process can take place only after a series of biochemical changes to the sperm occur in the female reproductive tract, collectively called capacitation. Spermatozoa can undergo spontaneous-acrosome reaction (sAR) before reaching the vicinity of the egg, preventing successful fertilization. Several mechanisms were shown to protect spermatozoa from undergoing sAR. Here we describe the involvement of the actin cross-linker, Ezrin in the mechanism that protects spermatozoa from sAR. Inhibition of Ezrin stimulates sAR and inhibits actin polymerization. Ezrin is highly phosphorylated/activated during the first hour of the capacitation process, and its phosphorylation rate is subsequently decreased. Ezrin phosphorylation depends on protein kinase A (PKA) and calmodulin kinase II (CaMKII) activities, and to some extent on phosphatidyl-inositol-4-kinase (PI4K) activity. Inhibition of these three kinases stimulates sAR, in which the effect of PI4K inhibition, but not PKA or CaMKII inhibition, can be reversed by increasing p-Ezrin using a phosphatase inhibitor. All together, we showed that three kinases mediate Ezrin activation during spermatozoa capacitation, leading to actin polymerization in a mechanism that prevents sAR.

A possible mechanism for the bactericidal effect of visible light.

Visible light at high intensity was found to kill bacteria while low-power light in the visible and near infrared region enhances bacterial proliferation. The present review summarizes evidence demonstrating that the mechanism of visible light- bacteria interaction involves reactive oxygen species (ROS) generation. The ROS are photo induced by bacterial endogenous photosensitizers. Phototoxic effects were found to involve induction of high amounts of reactive oxygen species (ROS) by the bacteria while low amounts of ROS may promote their proliferation. Intense blue light, preferably at 415nm, is better than red light for bacteria killing.

Photodynamic antimicrobial chemotherapy by liposome-encapsulated water-soluble photosensitizers.

Photodynamic antimicrobial chemotherapy is an alternative method for killing bacterial cells in view of the increasing problem of multi-antibiotic resistance. We examined the effect of three water-soluble photosensitizers (PhS): methylene blue (MB), neutral red (NR) and rose bengal (RB) on Gram-positive and Gram-negative bacteria. We compared the efficacy of PhS in their free form and encapsulated in liposomal formulations against various bacterial strains, and determined conditions for the effective use of encapsulated PhS. We found that all three PhS were able to eradicate the Gram-positive microbes Staphylococcus aureus and Sarcina lutea; and MB and RB were effective against St. epidermidis. In the case of the Gram-negative species, MB and RB were cytotoxic against the Shigella flexneri, NR-inactivated Escherichia coli and Salmonella para B, and BR was effective in killing Pseudomonas aeruginosa. None of the examined PhS showed activity against Klebsiella pneumoniae. MB and NR enclosed in liposomes gave a stronger antimicrobial effect than free PhS for all tested prokaryotes, whereas encapsulation of RB led to no increase in its activity. We suggest that encapsulation of PhS can increase the photoinactivation of bacteria.

Porin isolated from the outer membrane of Erwinia amylovora and its encoding gene.

A major Erwinia amylovora outer-membrane protein (Omp-EA) and the gene encoding for this protein (omp-EA) were isolated and characterized. The native Omp-EA protein forms a trimeric structure of approximately 114 kDa. This protein demonstrated high resistance to detergents such as SDS and octyl-glucopyranoside, but disaggregated to monomers with a molecular weight (MW) of approximately 39 kDa after heating at 95 degrees C for 10 minutes in sample buffer. The pore-forming ability of the oligomeric Omp-EA was determined by the liposome swelling assay, demonstrating that the oligomeric protein formed nonspecific channels with an exclusion limit of approximately 660 Da. On dissociation, the monomers did not exhibit pore-forming ability. The omp-EA gene was cloned and sequenced (GenBank Accession No. DQ184680). Sequence analysis revealed an open reading frame of 1152 bases. The deduced amino-acid sequence had 383 amino acids. The mature protein consisted of 362 amino acids and had a calculated MW of 39,210 Da. Multiple-sequence alignment of Omp-EA with other porins from the Enterobacteriaceae family revealed 51% to 63% identity. The first 16 amino acids from the N-terminal exhibited the highest identity (100%) to the porins OmpC, OmpF, and PhoE of Escherichia coli. Two methods were used to predict the secondary structure: APSSP2 and Hidden and Markov's model. The monomers of Omp-EA porin presented a topology of 16 transmembranal beta-strands. The area of the loops between the beta -strands was proposed. It is suggested that further research on the porin and its loops may be important for understanding the mechanism of E. amylovor to invade plant tissues.

Die-off of Cryptosporidium parvum in soil and wastewater effluents.

AIMS: To determine the effect of biotic and abiotic components of soil on the viability and infectivity of Cryptosporidium parvum, and evaluate the suitability of viability tests as a surrogate for oocyst infectivity under various environmental settings. METHODS AND RESULTS: The die-off of C. parvum in saturated and dry loamy soil was monitored over time by immunofluorescence assay (IFA) and PCR to estimate oocysts viability and by cell culture to estimate oocysts infectivity. Pseudomonas aeruginosa activity resulted in digestion of the outer layer of the oocysts, as demonstrated by loss of the ability to react in IFA. Whereas, P. aeruginosa activity did not affect the DNA amplification by PCR. A 1-log reduction in the oocysts infectivity was observed at 30 degrees C in distilled water and in saturated soil while oocysts viability was unchanged. Incubation for 10 days in dry loamy soil at 32 degrees C resulted in a 3-log(10) reduction in their infectivity while no change of oocysts viability was recorded. CONCLUSIONS: Under low temperature, C. parvum oocysts may retain their infectivity for a long time. Soil desiccation and high temperatures enhance the die-off rate of C. parvum. SIGNIFICANCE AND IMPACT OF THE STUDY: Previous die-off studies of C. parvum used viability tests that do not necessarily reflect the oocyst infectivity. Under low temperatures, there was an agreement observed between viability and infectivity tests and oocysts retained their infectivity for a long time. Desiccation and high temperatures enhance the loss of infectivity of C. parvum. The presented die-off data have significant implications on the management of wastewater reuse in warm environments.

An easy sonochemical route for the encapsulation of tetracycline in bovine serum albumin microspheres.

A one-step sonochemical process starting with the native BSA and tetracycline was employed to encapsulate the antibiotic drug in microspheres of BSA. The tetracycline loading studies showed that the maximum tetracycline loading capacity was found to be 65%. The antimicrobial activity of the tetracycline loaded in BSA microspheres was demonstrated on two bacterial strains that are sensitive to tetracycline.

Contribution of microbial activity to virus reduction in saturated soil.

Application of wastewater to soil may result in the contamination of groundwater and soil with pathogenic microorganisms and other biological and chemical agents. This study was performed to determine the antiviral microbial activity of soil saturated with secondary effluent. Low concentrations (0.05mg/ml) of protease pronase resulted in the inactivation of more than 90% of seeded Cox-A9 virus, whereas Poliovirus type 1, Hepatitis A virus (HAV) and MS2 bacteriophages were found to be insensitive to the enzyme activity. Exposure of Cox A9 virus to P. aeruginosa extracellular enzymes resulted in 99% inactivation of the seeded virus. Hepatitis A virus was found to be as sensitive as the Cox A9 virus, whereas Poliovirus 1 and MS2 were found to be insensitive to P. aeruginosa extracellular enzymatic activity. Furthermore, the time required for 99% reduction (T99) of Cox A9 and MS-2 Bacteriophage, at 15 degrees C, in soil saturated with secondary effluent was found to be 7 and 21 days, respectively. Faster inactivation was observed for MS2 and Cox A9 in soil saturated with secondary effluent incubated at 30 degrees C, T99 of 2 and 0.3 days, respectively. Although the concentration of the total bacterial count in the soil samples increased from 10(3) cfu/g to 10(5) cfu/g after 20 days of incubation at 30 degrees C, the proteolytic activity was below the detection level. The results of this study indicate that the virucidal effect of microbial activity is virus type dependent. Furthermore microbial activity in the soil material can be enhanced by the application of secondary effluent at higher temperature. The results also showed that MS2 bacteriophage can be used to predict viral contamination of soil and groundwater.

Photoinactivation of Acinetobacter baumannii and Escherichia coli B by a cationic hydrophilic porphyrin at various light wavelengths.

Photodynamic treatment by the cationic TMPyP photosensitizer was undertaken on the multiple antibiotic-resistant bacteria Acinetobacter baumannii and Escherichia coli. Total eradication of the bacterial cultures was determined immediately after initiation of illumination when these bacteria were treated with 5, 10, 15, 20-tetra (4-N methylpyridyl)porphine (TMPyP) at a concentration of 29.4 micromol/L and illuminated by blue, green, or red light. Total eradication of both bacteria was obtained also after treatment of bacterial cultures with 3.7 micromol/L TMPyP and illumination with blue light (400-450 nm). On the other hand, an 8- or 16- to 20-fold higher light intensity, respectively, was required for total eradication upon illumination with green (480-550 nm) or red light (600-700 nm). A 407-nm blue light only 7 and 9 joules/cm2, respectively, was needed for total eradication of both bacteria even at a concentration of 3.7 micromol/L TMPyP. X-ray-linked microanalysis demonstrated loss of potassium and a flood of sodium and chloride into the cells, indicating serious damage to the cytoplasmic membrane. Transmission electron microscopy (TEM) revealed structural changes and damage to the membrane of treated E. coli. In A. baumannii-treated cells, mesosomes and black dots that resemble aggregation of polyphosphate polymers could be seen. DNA breakage appeared only after a long period of illumination, when the bacterial cell was no longer viable. It can be concluded that cytoplasmic membrane damage and not DNA breakage is the major cause for bacterial death upon photosensitization.

Isolation of Vibrio vulnificus from sea water and sand along the Dan region coast of the Mediterranean.

The occurrence of Vibrio vulnificus incoastal sea water and sand was investigated. Samples (286 in toto) were taken during the period between November 1993 and July 1994. Ten V. vulnificus isolates (6.9%) were recovered from sea water and two isolates were recovered from sand (1.4%). The total isolation rate for this period was 4.2%. In a longer period of investigation, from June 1996 until June 1998 (24 months), 1,248 samples were taken and 205 V. vulnificus isolates were recorded (32.8%) in sea water while only 18 isolates in sand (2.9%). The total isolation rate was 17.9%. The monthly occurrence of this bacterial species in the various beaches surveyed demonstrated that V. vulnificus is more frequent during the months of July, August and September. The increase in the number of isolates during the past 2 years started as early as March and finished as late as October. Antibiotic sensitivity testing revealed that this species is sensitive to most antibiotics, except polymyxin B and colistin. The relatively high isolation rate of this bacterium from sea water may be dangerous to bathers, fishermen and divers with predisposed wounds.

Characterization of porins isolated from the outer membrane of Serratia liquefaciens.

A major outer membrane protein with an apparent molecular weight of 42 kDa was purified from Serratia liquefaciens grown on Brain Heart Infusion medium. The same protein was obtained when the cells were grown on a synthetic medium supplemented with 2% glucose. The amino acid composition of this protein revealed it to be hydrophilic. The pore-forming ability of the 42-kDa protein was determined by the liposome swelling assay. This assay demonstrated that the protein forms nonspecific channels with a diameter between 1.16 and 1.6 nm. An additional protein with a molecular weight of 47 kDa was obtained on synthetic medium supplemented with maltose. This protein exhibited specific pore-forming ability to maltose and maltodextrins, but was also permeable to other compounds, according to their size. When bacteria were grown on Nutrient Broth medium, two outer membrane proteins with molecular weights of 41 kDa and 42 kDa were produced by the bacteria. All three types of proteins represent monomers of respective oligomers. The monomers did not exhibit pore-forming ability when incorporated into liposomes. We, therefore, propose that the oligomer is the functional unit of a porin capable of forming permeability channels in the outer membrane of Serratia liquefaciens. These results indicate that S. liquefaciens contains several porins exhibiting specific osmoregulation or that are induced by a specific nutrient, where the 42-kDa outer membrane protein of this bacterium is certainly a major porin.

Characterization of porins isolated from the outer membrane of Vibrio damsela.

A protein oligomer with an approximate molecular weight of its 37-kDa monomer form was purified from the cell envelope fraction of Vibrio damsela cells. This oligomer exhibited strong porin activity when reconstituted into proteoliposomes with phosphatidyl choline. The functional properties for the 37-kDa protein suggest that it is a nonspecific or general porin, with an apparent pore size of 1.6 nm. This porin allows penetration of a variety of hydrophilic solutes according to their molecular mass. After electroelution, the oligomer was partially dissociated into monomers, whereas treatment with EDTA did not affect its dissociation. The monomers of the 37-kDa protein were not active in the reconstitution assay. The effect of culture media on the composition of the outer membrane protein of V. damsela was examined. Only one outer membrane protein with an apparent molecular weight of 37 kDa (37-kDa protein) was formed in cells grown in 3% NaCl-BHI broth and in 3% NaCl-nutrient broth with the addition of 2% glucose. Three outer membrane proteins, with apparent molecular weights of 37 kDa, 40 kDa, and 46 kDa, were produced in cells grown in 3% NaCl-nutrient broth. An additional outer membrane protein with an apparent molecular weight of 44 kDa (44-kDa protein) was found in cells grown in 3% NaCl-nutrient broth with the addition of 2% maltose. This protein was found to exhibit specificity to maltose derivatives. The results obtained in this study confirm the porin-like character of discussed proteins and give a basis for advanced study of those proteins.

Isolation and characterization of heat-modifiable proteins from the outer membrane of Porphyromonas asaccharolytica and Acinetobacter baumannii.

Active porins were isolated and purified from the outer membranes of the gram-negative anaerobic rod Porphyromonas asaccharolytica and the aerobic coccobacillus Acinetobacter baumannii. The porins from both bacteria appear to be monomers when isolated and purified. Both porins exhibited decreased mobility on SDS-PAGE after boiling for 10 min in the sample buffer. After heating, their molecular weight is estimated at 43 kDa while without heating they run as proteins with a molecular weight of approximately 37 kDa. Due to their characteristic heat-modifiability, these proteins were named HMP (heat-modifiable protein)-P. asaccharolytica and HMP-A. baumannii. Amino acid analysis revealed both porins to be hydrophilic proteins. These proteins have been shown to be active in transporting sugars when incorporated into liposomes. The permeability of both porins for L-arabinose was less than that produced by the porin of Escherichia coli B. Permeability to high molecular weight disaccharides was lower than for small monosaccharides. Western blot analysis did not reveal any antigenic cross reaction between HMP-A. baumannii and the HMP-P. asaccharolytica. The results obtained in this study confirm that although these heat-modifiable proteins are pore forming proteins and have similar activity they differ in their antigenicity.

Eradication of Acinetobacter baumannii by photosensitized agents in vitro.

The photodynamic effects of photosensitizers on Acinetobacter baumannii were studied. These Gram negative bacteria have recently been implicated in various infections, mainly acquired in hospitals. They have outstanding characteristics of multidrug high resistance to antimicrobial agents. The best photodynamic effect was obtained when A. baumannii cultures were treated with light activated deuteroporphyrin (Dp) at a concentration of 34 mumoles l-off and polymyxin nonapeptide (PMNP) at a concentration of 200 mumoles l-1. At these concentrations the culture in brain heart infusion (BHI) broth was found to be sterile after l h of treatment. Some inhibition was also obtained under the same conditions with Cd-texaphyrin (Cd-Tx) in the presence of PMNP. Treatment with various other photosensitizers in the presence of PMNP exhibited only marginal antibacterial activity. The cationic photosensitizer tetra-methylpyridyl porphine (TMPyP) did not exhibit any photodynamic effect on A. baumannii when illuminated during its growth in BHI broth. Bacteria grown in nutrient broth or suspended in saline and treated by TMPyP resulted in a significant photoinactivation by the sensitizer alone even in the absence of PMNP. It was found that a high concentration of the proteins present in BHI or in serum prevent TMPyP from acting as a photosensitizer against A. baumannii. Bovine serum albumin at the same high protein concentration prevents Dp (in the presence of PMNP) to act as a photosensitizer. The anionic photosensitizer tetra-sulfonatophenyl porphine (TPPS4) did not show any photodynamic effect in high or low protein media. In this study it was found that despite the high resistance of the Acinetobacter baumannii to antibiotics, these bacteria can be significantly photoinactivated by treatment with either Dp + PMNP or TMPyP in low protein content environments. When the protein concentration is high photoinactivation efficiency depends on the type of protein present in the medium.

The use of porphyrins for eradication of Staphylococcus aureus in burn wound infections.

The assessment of deuteroporphyrin-hemin complex as an agent for the treatment of burn wounds infected with a multiple-drug resistant strain of Staphylococcus aureus was performed. The effect of the porphyrin on the survival of the infectious bacteria was first assayed in culture, and later tested as well in a burned infected animal model. The addition of deuteroporphyrin and hemin, separately or together (as a complex) to a growing culture of S. aureus was monitored during 8 hours. It was found that deuteroporphyrin alone was strongly bactericidal only after photosensitization. On the other hand, hemin alone was moderately bactericidal but light independent. A combination of both deuteroporphyrin and hemin was extremely potent even in the dark and did not require illumination to eradicate the bacteria. The in vivo experiments by application of the above porphyrins in combination to infected burn wounds in guinea pigs was an effective way to reduce dramatically the contaminating S. aureus. Reduction of more than 99% of the viable bacteria was noted after the porphyrin mixture was dropped on the eschar or injected into the eschar, an effect that lasted for up to 24 hours. The deuteroporphyrin-hemin complex may be suggested as a new bactericidal treatment of S. aureus infected burns since it was found to be a potent and promising anti-Staphylococcal agent.

The dynamics of inflammation of the anterior eye in a novel experimental model for hypersensitivity.

A novel immunologically provoked inflammatory process was studied in guinea pigs. The animals were immunized by i.p. injections of ovalbumin (OA) suspended in Freund's complete adjuvant and challenged by the application of OA into the conjunctival sac of one eye. An inflammatory reaction was seen a few minutes after provocation and lasted normally for 4-7 days. The process was characterized by early damage to the epithelial layer which was partly detached in small flakes; an intense tearing with the tear fluid soon turning mucous and then purulent; vasodilation in the bulbar conjunctiva, in particular towards the limbal region; margination and emigration of polymorphonuclear, and to a lesser extent, eosinophil, leucocytes which migrated towards and infiltrated the surface epithelial layer. Subsequently, the dominant cell type infiltrating the submucosa was lymphocytes. Later, opacity of the cornea occurred, probably due to oedema and neovascularization of the stroma progressing centrally from the periphery. When the antigenic challenge was repeated, thickening of the conjunctival mucosa, and neoformation of collagen bundles in the submucosa led to the swelling of the upper lids. The facets of this inflammatory trauma may not fit easily into any of the classical types of hypersensitivity. Rather, it may combine features of several of them, at least type 1 and type 4. This syndrome shows several features similar to those of human vernal keratoconjunctivitis.

Structure-activity relationship of porphines for photoinactivation of bacteria.

The antibacterial photodynamic effects of uncharged (o-tetrahydroxyphenyl porphine [THPP], m-THPP and p-THPP), cationic (5,10,15,20-tetra[4-N-methylpyridyl]porphine [TMPyP]) and anionic (5,10,15,20-tetra[4-sulfonatophenyl porphine] [TPPS4]) porphines on Staphylococcus aureus and Escherichia coli bacteria inactivation were examined. The results show that uncharged porphines provoked antibacterial photodynamic activity on S. aureus, and also on E. coli in the presence of the membrane-disorganizing peptide polymixin B nonapeptide (PMNP). The TMPyP compound was highly photoactive toward gram-positive bacteria but only marginally effective on gram-negative cells, whereas TPPS4 showed no activity on either gram-positive or gram-negative bacteria. The photoactivity of TMPyP is due to the electrostatic attraction between the positively charged sensitizer molecule and the negatively charged membrane of the gram-positive target cells. For TPPS4, the inactivity toward gram-positive bacteria is due to electrostatic repulsion between the charged sensitizer molecule and the cell membrane. For gram-negative bacteria, the inactivity is conceivably due to preferential (electrostatic) binding to the positively charged PMNP, which is an adjuvant for membrane disorganization, but has no effect on cell viability. For hydrophobic sensitizers, the photoactivity depends on the state of aggregation. The extent of deaggregation of the different THPP isomers was determined by fluorescence measurements of bound sensitizers and could be positively correlated with their photoinactivation capacity. We conclude that the structure-activity relationships of these porphines are affected by their net charge and by aggregation.

A 28,000 mol. wt toxin from Bacillus thuringiensis israelensis induces cation transport in rat muscle cultures.

The mechanism by which the Bacillus thuringiensis israelensis (Bti) 28,000 mol. wt toxin exerts its effect on mature muscle cultures was examined. The toxin inhibited Na+/K(+)-ATPase activity as revealed by 86Rb influx. A 50% inhibition of Na+/K(+)-ATPase activity was obtained with 0.2 microgram/ml of the toxin. The inhibition was time and dose dependent, and it was reversible with low doses of the toxin (up to 0.2 microgram/ml. A considerable release of 86Rb was obtained by doses greater than 0.2 microgram/ml. The 86Rb release was also time and dose dependent. This effect is probably non-specific, since 45Ca influx is also accelerated by toxin-treated cultures. Pre-incubation of the toxin with phosphotidylserine (PS) antagonized the toxin. It is concluded that the toxin is a hydrophobic protein which interacts with the membrane. In low doses this interaction reduces the activity of the sodium pump and in high doses it causes non-specific permeability of the sarcolemma.

Spectroscopy and photosensitization of sapphyrins in solutions and biological membranes.

A spectroscopic and photophysical study of three new sapphyrin molecules is presented. The sapphyrin backbone that was derivatized to make them water soluble possesses an absorption band around 700 nm, a desired property for biological photosensitization. We studied the absorption and fluorescence spectra, from which evidence for aggregation in solvents of different polarities was obtained. The extent of aggregation is correlated with the nature of the attached moiety. The absolute quantum yields of singlet oxygen production were measured, with 1,3-diphenyl isobenzofuran as a model target, and were 0.13-0.18 in ethanol. The binding constants to liposomes and to cells were determined spectroscopically and were found to correspond to the hydrophobicities of the compounds, with an additional effect, ascribed to the sugar moiety, which was found in the case of one of the sapphyrins. The efficiency of photodamage to Staphylococcus aureus by sapphyrins and hematoporphyrin was equivalent, on the basis of cells killed per microgram of sensitizer in the incubation mixture.

Biochemical and morphological changes in rat muscle cultures caused by 28,000 mol. wt toxin of Bacillus thuringiensis israelensis.

The 28,000 mol. wt protein of Bacillus thuringiensis israelensis showed a high degree of toxicity to rat muscle in culture. Application of 1 microgram/ml to the culture medium completely inhibited cell fusion. Reversibility of this effect was demonstrated by replacement of the culture medium with fresh medium, and the consequence was that cell fusion was resumed. When differentiated myotubes were treated with 1 microgram/ml of the toxin, the spontaneous contractile activity was abolished within 20 min. Cytotoxic effects were observed 1 hr after treatment was initiated, as manifested by creatine kinase (CK) release to the medium. Two hours after toxin was applied to the muscle culture, the myotubes were deteriorated whereas the mononucleated cells were not affected. Six or 7-day-old cultures which were treated by 1 microgram/ml of 28,00 mol. wt toxin revealed a change in the levels of Na+ and K+ within the fibres as analysed by X-ray microanalysis (XRMA). Preincubation of the toxin for 20 min with phospholipids before application to the cells reduced the cytotoxic effect. Phosphatidylinositol and phosphatidylserine were the most efficient inhibitors, whereas phosphatidylcholine, sphingomyelin and phosphatidylethanolamine were less effective in protecting cultures from the cytotoxic effects of the 28,000 mol. wt protein.