Experimental Infections with Mycoplasma agalactiae Identify Key Factors Involved in Host-Colonization

Mechanisms underlying pathogenic processes in mycoplasma infections are poorly understood, mainly because of limited sequence similarities with classical, bacterial virulence factors. Recently, large-scale transposon mutagenesis in the ruminant pathogen Mycoplasma agalactiae identified the NIF locus, including nifS and nifU, as essential for mycoplasma growth in cell culture, while dispensable in axenic media. To evaluate the importance of this locus in vivo, the infectivity of two knock-out mutants was tested upon experimental infection in the natural host. In this model, the parental PG2 strain was able to establish a systemic infection in lactating ewes, colonizing various body sites such as lymph nodes and the mammary gland, even when inoculated at low doses. In these PG2-infected ewes, we observed over the course of infection (i) the development of a specific antibody response and (ii) dynamic changes in expression of M. agalactiae surface variable proteins (Vpma), with multiple Vpma profiles co-existing in the same animal. In contrast and despite a sensitive model, none of the knock-out mutants were able to survive and colonize the host. The extreme avirulent phenotype of the two mutants was further supported by the absence of an IgG response in inoculated animals. The exact role of the NIF locus remains to be elucidated but these data demonstrate that it plays a key role in the infectious process of M. agalactiae and most likely of other pathogenic mycoplasma species as many carry closely related homologs.     

Larvicidal activity of essential oils from Brazilian plants against Aedes aegypti L

Aedes aegypti L. is the major vector of dengue fever, an endemic disease in Brazil. In an effort to find effective and affordable ways to control this mosquito, the larvicidal activities of essential oils from nine plants widely found in the Northeast of Brazil were analyzed by measurement of their LC50. The essential oils were extracted by steam distillation and their chemical composition determined by GL-chromatography coupled to mass spectroscopy. The essential oils from Cymbopogon citratus and Lippia sidoides, reported in the literature to have larvicidal properties against A. aegypti, were used for activity comparison. The results show that Ocimum americanum and Ocimum gratissimum have LC50 of 67 ppm and 60 ppm respectively, compared to 63 ppm for L. sidoides and 69 ppm for C. citratus. These results suggest a potential utilization of the essential oil of these two Ocimum species for the control of A. aegypti.     

Effect of activated charcoal on multiplication of African yam (Dioscorea cayenensis-rotundata) nodal segments using a temporary immersion bioreactor (RITA®)

In this study, we compared the growth of Dioscorea cayenensis-rotundata (African yam) nodal segments, using semisolid medium in test tubes and liquid medium in 1-L Recipient for Automated Temporary Immersion (RITA^®) temporary immersion bioreactors (TIB), and the application of various culture parameters. The addition of activated charcoal (AC) had a positive effect on the growth of nodal segments, both in semisolid medium and in liquid medium in RITA^® bioreactors. After 2 mo culture in the presence of AC, plantlets were 6.4–6.6 cm long compared to 3.2–3.8 cm in absence of AC, with no significant difference observed between the culture systems. In the range of inoculation densities tested (5–20 nodal segments per RITA^® bioreactor), there was no effect on the number of buds produced per nodal segment, the moisture content of plantlets (fresh weight basis), or on net fresh weight gain. By contrast, the individual leaf surface area of plantlets decreased in line with increasing inoculation density. Among the range of benzylaminopurine (BAP) concentrations tested (0–17.6 μM), 0.44 μM induced the highest number of buds (3.8 buds per nodal segment) in the TIB. However, comparable numbers of buds could be produced with media devoid of BAP, either by increasing the frequency of 1-min daily immersion cycles in RITA^® bioreactors from one every 12 h to one every 4 h or by using semisolid medium containing AC.     

Synthetic Aurones: New Features for Schistosoma mansoni Therapy

In this work, two synthetic aurones revealed moderate schistosomicidal potential in in vitro and in vivo assays. Aurones ( 1 ) and ( 2 ) promoted changes in tegument integrity and motor activity, leading to death of adult Schistosoma mansoni worms in in vitro assays. When administered orally (two doses of 50 mg/kg) in experimentally infected animals, synthetic aurones ( 1 ) and ( 2 ) promoted reductions of 56.20 % and 57.61 % of the parasite load and stimulated the displacement towards the liver of the remaining adult worms. The oogram analysis revealed that the treatment with both aurones interferes with the egg development kinetics in the intestinal tissue. Seeking an action target for compounds ( 1 ) and ( 2 ), the connection with NTPDases enzymes, recognized as important therapeutic targets for S. mansoni, was evaluated. Molecular docking studies have shown promising results. The dataset reveals the anthelmintic character of these compounds, which can be used in the development of new therapies for schistosomiasis.     

Interaction of plasma proteins with negatively charged sites on the pulmonary capillary endothelium of the rat

Summary The endothelial glycocalyx, a polyanionic structure which may regulate the passage of solutes and water through the endothelium, readily binds cationic ferritin (CF). In normal, nonexchange-transfused rats, however, only 7.5% and 6.0% of the luminal plasma membrane and 7.5% and 5.0% of vesicle diaphragms on the thick and thin side of pulmonary capillaries, respectively, bound cationic ferritin. With the graded removal of circulating proteins by exchange transfusion with fluorocarbon emulsion, up to 89 and 82% of the luminal surface, and 76 and 73% of vesicle diaphragms on the thick and thin sides, respectively, bound CF. Although the extent of binding on the thin side was consistently less than on the thick side, the difference was not statistically significant. The extensive binding of CF to the glycocalyx in totally exchange-transfused rats was completely reversible upon addition of lyophilized rat serum protein to the perfusate. These data suggest that in vivo anionic sites of the endothelial glycocalyx are partially masked by adsorbed plasma proteins.     

Knockdown of occludin expression leads to diverse phenotypic alterations in epithelial cells

The function of occludin (Occ) in the tight junction is undefined. To gain insight into its role in epithelial cell biology, occludin levels in Madin-Darby canine kidney II cells were suppressed by stably expressing short interfering RNA. Suppression of occludin was associated with a decrease in claudins-1 and -7 and an increase in claudins-3 and -4. Claudin-2 levels were unaffected. The tight junction "fence" function was not impaired in suppressed Occ (Occ–) clones, as determined by BODIPY-sphingomyelin diffusion in the membrane. The most striking changes were those related to control of the cytoskeleton and the "gate" function of tight junctions. A reduced ability of Occ– clones to extrude apoptotic cells from the monolayers suggested that neighbors of apoptotic cells either failed to sense their presence or were unable to coordinate cytoskeletal activity necessary for their extrusion. To further test the extent to which actin cytoskeletal activity depends on the presence of occludin, Occ– and Occ+ monolayers were depleted of cholesterol. Previous studies showed that cholesterol depletion is associated with reorganization of the actin cytoskeleton and a fall in transepithelial electrical resistance. In contrast to control Occ (Occ+) cells, transepithelial electrical resistance did not fall significantly in cholesterol-depleted Occ– monolayers and they failed to generate Rho-GTP, one of the signaling molecules involved in regulating the actin cytoskeleton. While steady-state transepithelial electrical resistance was similar in all clones, tight junction permeability to mono- and divalent inorganic cations was increased in Occ– monolayers. In addition, there was a disproportionately large increase in permeability to monovalent organic cations, up to 6.96 Å in diameter. Chloride permeability was unaffected and there was little change in mannitol flux. The data suggest that occludin transduces external (apoptotic cells) and intramembrane (rapid cholesterol depletion) signals via a Rho signaling pathway that, in turn, elicits reorganization of the actin cytoskeleton. Impaired signaling in the absence of occludin may also alter the dynamic behavior of tight junction strands, as reflected by an increase in permeability to large organic cations; the permeability of ion pores formed of claudins, however, is less affected. tight junction; occludin; Rho-GTP     

Ovarian cycle of southern brown howler monkey (<Emphasis Type="Italic">Alouatta guariba clamitans</Emphasis>) through fecal progestin measurement

The ovarian cycle in howler monkeys (genus Alouatta) has beean investigated through several biological parameters (ranging between 16.3 and 29.5 days); however, no data exist concerning the ovarian activity of the southern brown howler monkey (Alouatta guariba clamitans). This study aimed to describe the ovarian cycle of A. g. clamitans by profiling fecal progestin concentrations. Over 20 weeks, fecal samples of eight captive adult females of A. g. clamitans were collected. The collections were made at dawn, 5 days a week, and the samples were frozen immediately following collection. Next, they were dried, pulverized and hormonal metabolites were extracted to determine progestin concentrations by enzyme immunoassay. Of the 758 samples tested, the mean concentration of fecal progestins was 2866.40 ± 470.03 ng/g of dry feces, while the mean concentration at baseline was 814.47 ± 164.36 ng/g of dry feces. Among the eight females, one showed no ovarian cyclicity and three presented periods of probable absence of cyclicity and low progestin concentrations. A mean duration of 16 ± 0.52 days was observed for the 35 cycles studied. The interluteal phase lasted 4 ± 0.37 days on average, with a mean concentration of fecal progestins of 467.98 ± 29.12 ng/g of dry feces, while the luteal phase lasted 11 ± 0.50 days, with a mean concentration of 4283.27 ± 193.31 ng/g of dry feces. Besides describing the characteristics of the ovarian cycle, possible causes for the low concentrations of fecal progestins and periods of absence of cyclicity are also discussed.     

Purified mouse egg zona pellucida glycoproteins polymerize into homomeric fibrils under non-denaturing conditions

The mouse egg's zona pellucida (ZP) is composed of three glycoproteins, called ZP1, ZP2, and ZP3, that migrate as relatively broad, single bands on SDS-PAGE. The glycoproteins are organized within the ZP as a network of long interconnected fibrils that exhibit a structural periodicity. Here, ZP2 and ZP3 were purified by HPLC to homogeneity and analyzed by Blue Native- (BN-) PAGE and transmission electron microscopy (TEM), as well as by SDS-PAGE. As opposed to SDS-PAGE, BN-PAGE, and TEM permit analysis of ZP2 and ZP3 under non-denaturing conditions. ZP2 and ZP3 migrate on BN-PAGE, not as single bands, but as several discrete oligomers that give rise to larger structures which remain at the origin of the gel. Consistent with this, ZP2 and ZP3 are visualized by TEM as long interconnected fibrils that consist of contiguous beads. Therefore, under non-denaturing conditions both purified ZP2 and ZP3 polymerize into higher order structures. These findings are of interest since purified ZP3 inhibits binding of mouse sperm to eggs and induces sperm to undergo the acrosome reaction in vitro. Results presented here suggest that these biological effects of ZP3 are due to binding of homomeric fibrils of ZP3 to sperm.     

Development and Host Compatibility of Plasmids for Two Important Ruminant Pathogens,Mycoplasma bovis and Mycoplasma agalactiae

Mycoplasma bovis is a cause of pneumonia, mastitis, arthritis and otitis media in cattle throughout the world. However, despite its clinical significance, there is a paucity of tools to genetically manipulate it, impeding our capacity to further explore the molecular basis of its virulence. To address this limitation, we developed a series of homologous and heterologous replicable plasmids from M. bovis and M. agalactiae. The shortest replicable oriC plasmid based on the region downstream of dnaA in M. bovis was 247 bp and contained two DnaA boxes, while oriC plasmids based on the region downstream of dnaA in M. agalactiae strains 5632 and PG2 were 219 bp and 217 bp in length, respectively, and contained only a single DnaA box. The efficiency of transformation in M. bovis and M. agalactiae was inversely correlated with the size of the oriC region in the construct, and, in general, homologous oriC plasmids had a higher transformation efficiency than heterologous oriC plasmids. The larger pWholeoriC45 and pMM21-7 plasmids integrated into the genomic oriC region of M. bovis, while the smaller oriC plasmids remained extrachromosomal for up to 20 serial passages in selective media. Although specific gene disruptions were not be achieved in M. bovis in this study, the oriC plasmids developed here could still be useful as tools in complementation studies and for expression of exogenous genes in both M. bovis and M. agalactiae.