Hemolytic and hemoxidative activities in Mycoplasma penetrans

Mycoplasma penetrans is a newly isolated Mollicute from the urine of patients infected with human immunodeficiency virus that demonstrates the capacity to adhere to and invade human cells. A previous report, based on assays with mouse red blood cells (RBCs), indicated that M. penetrans lacked hemolytic activity. In our studies, we incubated different isolates of M. penetrans with various RBC species and observed hemolytic zones surrounding individual mycoplasma colonies. All M. penetrans strains displayed hemolysis after 2 to 3 days of incubation. Hemolytic activity diffused from single colonies, eventually causing complete lysis. Hemolysis was most pronounced with sheep RBCs, followed by horse, chicken, and human cells. Furthermore, hemolytic activity was demonstrable in both intact mycoplasma cell preparations and spent culture supernatant. However, unlike intact mycoplasmas, the hemolytic activity in the supernatant was dependent on the reducing agent, cysteine. In addition to hemolysis, a brown precipitate was closely associated with mycoplasma colonies, suggesting oxidation of hemoglobin. Absorption spectra indicated that hemoglobin was oxidized to methemoglobin, and the addition of catalase demonstrated H(2)O(2)-mediated hemoxidation. Other experiments suggested that hemoxidation enhanced total hemolysis, providing the first evidence of both hemolytic and hemoxidative activities in M. penetrans.     

Abdominal vagotomy attenuates interleukin-1β-induced nitric oxide release in the paraventricular nucleus region in conscious rats

Nitric oxide (NO) has recently been shown to modulate the hypothalamic–pituitary–adrenal axis response to interleukin-1β (IL-1β). We measured levels of nitrite (NO₂⁻) and nitrate (NO₃⁻) in the hypothalamic paraventricular nucleus (PVN) region using an in vivo brain microdialysis technique in conscious rats. Intraperitoneally administered IL-1β produced a significant increase in both NO₂⁻ and NO₃⁻ levels in the PVN region. We also examined the possible involvement of the abdominal vagal afferent nerves in this effect. In abdominal-vagotomized rats, the increase was significantly attenuated compared to that in sham-operated rats. Our results suggest that the abdominal vagal afferent nerves are involved in intraperitoneally administered IL-1β-induced NO release in the PVN region.     

Flowering in bursting bubbles with viscoelastic interfaces

The lifetime of bubbles, from formation to rupture, attracts attention because bubbles are often present in natural and industrial processes, and their geometry, drainage, coarsening, and rupture strongly affect those operations. Bubble rupture happens rapidly, and it may generate a cascade of small droplets or bubbles. Once a hole is nucleated within a bubble, it opens up with a variety of shapes and velocities depending on the liquid properties. A range of bubble rupture modes are reported in literature in which the reduction of a surface energy drives the rupture against inertial and viscous forces. The role of surface viscoelasticity of the liquid film in this colorful scenario is, however, still unknown. We found that the presence of interfacial viscoelasticity has a profound effect in the bubble bursting dynamics. Indeed, we observed different bubble bursting mechanisms upon the transition from viscous-controlled to surface viscoelasticity-controlled rupture. When this transition occurs, a bursting bubble resembling the blooming of a flower is observed. A simple modeling argument is proposed, leading to the prediction of the characteristic length scales and the number and shape of the bubble flower petals, thus paving the way for the control of liquid formulations with surface viscoelasticity as a key ingredient. These findings can have important implications in the study of bubble dynamics, with consequences for the numerous processes involving bubble rupture. Bubble flowering can indeed impact phenomena such as the spreading of nutrients in nature or the life of cells in bioreactors.

When residing in Newtonian fluids, bubble rupture proceeds with features that are shown in Fig. 1. Different dynamics are observed depending on the capillary number, Ca=u η/γ, where u is the experimentally measured characteristic retraction speed of the film, η is the liquid viscosity, and γ is the surface tension between the liquid and gas, and the Reynolds number, Re=ρuRbubble/η, where ρ is the liquid density, and Rbubble is the bubble radius (1–7). Fig. 1 A–D report observed bubble ruptures in the different regimes previously discussed in the literature: 1) for Re≪1, viscous forces are larger than inertial and surface forces, resulting in a very slow hole opening (8) (Fig. 1A); 2) when Re>1, the hole opens up much more quickly (9), and a toroidal rim (Fig. 1B) is subjected to an azimuthal instability, which leads to fingering and, possibly, jetting (Fig. 1 B–D), depending on the corresponding Ca value. If Ca≫1, the rim is stable and folds upward (10); otherwise, inertial instabilities break it into pieces with a characteristic length scale, d=Rbubbleh, where h is the film thickness (11).Open in a separate windowFig. 1.Bubble bursting dynamics. (A) Low Re. (B and C) High Ca and high Re. (D) Low Ca and high Re. (E) Low Ca and high Re with intermediate interfacial viscoelasticity (20 mg/mL of BSA). (F) Low Ca and high Re with high interfacial viscoelasticity (50 mg/mL of BSA). (G) Bubble rupture as functions of time for different concentrations of BSA. Increasing the concentration of BSA leads to an increase in the surface viscoelasticity, and the bubble bursting dynamics change. The concentration of 20 mg/mL is identified as the limit above which flowering occurs. The number of petals, 10 at 20 mg/mL, decreases to 5 when the concentration of BSA is increased to 50 mg/mL. The bursting time (from the puncture to complete film retraction) increases when BSA is added, and it changes from 1.5 ms at 0.1 mg/mL to 2.7 ms at 50 mg/mL.Here, we report evidence of the effect of surface viscoelasticity in bubble rupture dynamics. Surface viscoelasticity is adjusted by adding a surface-active material to the bulk and modulating its concentration and chemistry. We chose bovine serum albumin (BSA) proteins (12, 13), as they are known to form highly viscoelastic surface layers. At low concentrations, BSA molecules are adsorbed at the air/water interface with their major axis parallel to the surface. No protein denaturation occurs, and the molecules retain their globular conformation. As the concentration of BSA increases, a primary monolayer achieves full surface coverage, and a secondary monolayer appears, extending into the aqueous phase (14, 15). Adsorbed protein molecules are connected by interprotein contacts forming an interconnected network within the adsorbed layers (16). Upon compression, globular proteins, such as BSA, respond as deformable spheres, thereby being capable of storing elastic energy (yielding high storage moduli) (17). Many examples can be found in the literature in which the addition of surfactants to the protein solution can drastically change surface properties, giving an unlimited variety of model systems to achieve desired surface properties with a straightforward tuning of the surfactant concentration (18).     

Multisite Study of Cryptosporidiosis in Children with Diarrhea in India

Cryptosporidium spp., a common cause of diarrhea in children, were investigated in the first multisite study in India. Diarrheal stools from hospitalized children aged <5 years from Delhi, Trichy, and Vellore were analyzed by microscopy, PCR-restriction fragment length polymorphism (RFLP), and/or sequencing at the small-subunit (SSU) rRNA and Cpgp40/15 loci for species determination and subgenotyping, respectively. Seventy of 2,579 (2.7%) children, 75% of whom were <2 years old, had cryptosporidial diarrhea as determined by microscopy. Genotyping and subgenotyping showed that Cryptosporidium hominis was the most commonly identified species (59/67 children), and subgenotypes Ie, Ia, Ib, and Id were common in all centers. A novel C. parvum subgenotype, IIn, was identified in Vellore. Meteorological analysis revealed a higher rate of cryptosporidial positivity during hotter and drier weather in Delhi.Cryptosporidium spp. are an important cause of endemic parasitic diarrhea in children in developing countries. In addition to causing symptoms associated with watery diarrhea, vomiting, and weight loss, early childhood cryptosporidiosis has been shown by studies to be associated with subsequent faltering of growth (reviewed in reference 11). Cryptosporidium hominis and C. parvum cause the majority of infections in children in developing countries, with C. hominis predominating and occasional reports of infection with zoonotic species such as C. felis, C. canis, C. meleagridis, and C. muris (30). C. hominis infection has been found to be associated with greater levels of oocyst shedding (4) and longer durations of oocyst shedding (31) and diarrhea (15) than C. parvum infection. In a recent community-based study in Vellore, we found increased levels of severity of diarrhea in C. hominis-infected children compared to the levels observed in children infected with other species (1).Cryptosporidium spp. have been classified into several distinct subgenotypes based on extensive polymorphisms in the Cpgp40/15 (also referred to as GP60) locus by use of PCR-restriction fragment length polymorphism (RFLP) or sequencing of PCR products (reviewed in reference 30).A number of studies from India have reported Cryptosporidium spp. in diarrheal stool samples from children, with positivity rates of up to nearly 20% (17) and asymptomatic infection rates of up to 10% (19), using stool microscopy for detection. However, only three studies have used molecular techniques for identification of cryptosporidiosis in children in India (9, 13, 22), suggesting that the actual infection rates may be significantly higher. In a previous hospital-based study in Vellore, we that found that PCR (15.2%) identified more than 3 times the number of cases of cryptosporidial diarrhea than microscopy (4.4%) (2). The aim of the present study was to identify the Cryptosporidium species and Cpgp40/15 subgenotypes associated with cryptosporidial diarrhea in hospitalized children from 3 centers in the country, since no studies have examined cryptosporidiosis using the same methods in more than one location.     

Neonatal thrombosis

Neonatal thrombosis is a serious event that can cause mortality or result in severe morbidity and disability. The most important risk factor for the development of thrombosis during the neonatal period is the presence of an indwelling central line and consequently the vessels involved tend to be those most frequently used for catheterization. Other documented risk factors for the development of neonatal thrombosis include asphyxia, septicemia, dehydration, maternal diabetes and cardiac disease. Main laboratory findings for the diagnosis of hypercoagulable states, include shortened aPTT, decreased levels of inhibitors (AT III, Protein C and Protein S), increased resistance to activated protein C, defective fibrinolysis (basal and after stimuli), increased levels of clotting factors (fibrinogen, factor VII, factor VIII, etc.), increased and/or hyperactive platelets, increased whole blood and/or plasma viscosity, Antiphospholipid antibodies and presence of prothrombotic molecular defects like FV Leiden, P20210 and MTHFR. Approximately 4% and 2% respectively of Caucasians are heterozygous for these gene defects. Their causative role in neonatal thrombosis is unknown but they may have a contributory role in the pathogenesis of thrombosis in neonates.     

Vitamin C-induced loss of redox-dependent viability in lung microvascular endothelial cells

Recent clinical trials have shown that vitamin C, at pharmacological concentrations (milligram to approximately gram), upon infusion into circulation, modulates vasodilation and vascular tone in humans. This also results in the elevated concentrations of vitamin C in circulation in the millimolar range. Here, it was hypothesized that vitamin C at pharmacological concentrations (millimolar) would induce oxidative stress and cause loss of redox-dependent cell viability in vascular endothelial cells (ECs). To test the hypothesis, bovine lung microvascular ECs (BLMVECs) in monolayer cultures were exposed to vitamin C (0-10 mM) for different time periods (0-2 h). Electron paramagnetic resonance spectroscopy revealed the intracellular formation of ascorbate free radical in a dose- and time-dependent fashion. Vitamin C also induced formation of intracellular reactive oxygen species in a dose-dependent fashion. It was observed that vitamin C induced morphological alterations and loss of cell viability in a dose- and time-dependent fashion, as measured by light microscopy and Alamar Blue redox cell viability assay, respectively. Vitamin C analogues failed to induce such changes. Vitamin C depleted cellular GSH levels in a dose-dependent fashion, suggesting that vitamin C altered thiol-redox status in BLMVECs. Antioxidants, intracellular iron chelator, and catalase protected cells against vitamin C-induced loss of redox-dependent cell viability, confirming the role of hydrogen peroxide and iron during redox cycling of vitamin C. These results, for the first time in detail, established that vitamin C at pharmacological doses induced oxidative stress and loss of redox-dependent cell viability in microvascular ECs.     

Lysophosphatidic acid triggers calcium entry through a non-store-operated pathway in human neutrophils

Lysophosphatidic acid (LPA) is a bioactive lipid, which is structurally similar to sphingosine 1-phosphate (S1P) and which can mobilize Ca2+ in multiple cell types. We recently showed that S1P induces Ca2+ entry directly through store-operated Ca2+ entry (SOCE) channels in human polymorphonuclear neutrophils (PMN). We therefore examined the mechanisms by which LPA induces intracellular Ca2+ mobilization in PMN. External application of low micromolar LPA caused dose-dependent Ca2+ influx without releasing Ca2+ stores, whereas G-protein-coupled (GPC) LPA receptors respond to nanomolar LPA. Additive Ca2+ influx by LPA compared with 100 nM ionomycin-induced Ca2+ influx suggests that LPA-induced Ca2+ influx does not pass through SOCE channels. Ca2+ influx was resistant to inhibition of Gi/o by pertussis toxin, of phospholipase C by U73122, and of G12/13/Rho by Y27632, all demonstrating GPC receptor independence. This Ca2+ influx was inhibited by Gd3+, La3+, Zn2+, or MRS1845 but not by Ni2+ or the sphingosine kinase inhibitor dimethylsphingosine. In addition, we found that LPA has no effect on neutrophil chemotaxis; however, it has stimulatory effects on neutrophil respiratory burst in a dose-response manner. These findings suggest that LPA-induced Ca2+ influx in PMN occurs through a mechanism other than SOCE channels, independent of Ca2+ store-depletion and S1P synthesis, and that the characteristics of LPA-induced Ca2+ influx are similar to those of S1P-induced influx in terms of sensitivity to inorganic inhibitors. Unlike S1P, LPA has stimulatory effects on neutrophil respiratory burst.