Molecular hydrogen promotes retinal vascular regeneration and attenuates neovascularization and neuroglial dysfunction in oxygen-induced retinopathy mice.

BACKGROUND: Retinopathy of Prematurity (ROP) is a proliferative retinal vascular disease occurring in the retina of premature infants and is the main cause of childhood blindness. Nowadays anti-VEGF and retinal photocoagulation are mainstream treatments for ROP, but they develop a variety of complications. Hydrogen (H2) is widely considered as a useful neuroprotective and antioxidative therapeutic method for hypoxic-ischemic disease without toxic effects. However, whether H2 provides physiological angiogenesis promotion, neovascularization suppression and glial protection in the progression of ROP is largely unknown.This study aims to investigate the effects of H2 on retinal angiogenesis, neovascularization and neuroglial dysfunction in the retinas of oxygen-induced retinopathy (OIR) mice. METHODS: In this study, mice that were seven days old and either wild-type (WT) or Nrf2-deficient (Nrf2-/-) were exposed to 75% oxygen for 5 days and then returned to normal air conditions. Different stages of hydrogen gas (H2) inhalation were administered. Vascular obliteration, neovascularization, and blood vessel leakage were analyzed and compared. To count the number of neovascularization endothelial nuclei, routine HE staining of retinal sections was conducted. Immunohistochemistry was performed using DyLight 594 labeled GSL I-isolectin B4 (IB4), as well as primary antibodies against proliferating cell nuclear antigen (PCNA), glial fibrillary acidic protein (GFAP), and Iba-1. Western blots were used to measure the expression of NF-E2-related factor 2 (Nrf2), vascular endothelial growth factor (VEGF), Notch1, Dll4, and HIF-1α. Additionally, the expression of target genes such as NQO1, HO-1, Notch1, Hey1, Hey2, and Dll4 was measured. Human umbilical vein endothelial cells (HUVECs) treated with H2 under hypoxia were used as an in vitro model. RT-PCR was used to evaluate the mRNA expression of Nrf2, Notch/Dll4, and the target genes. The expression of reactive oxygen species (ROS) was observed using immunofluorescence staining. RESULTS: Our results indicate that 3-4% H2 does not disturb retinal physiological angiogenesis, but ameliorates vaso-obliteration and neovascularization in OIR mice. Moreover, H2 prevents the decreased density and reverses the morphologic and functional changes in retinal astrocytes caused by oxygen-induced injury. In addition, H2 inhalation reduces microglial activation, especially in the area of neovascularization in OIR mice. H2 plays a protective role in vascular regeneration by promoting Nrf2 activation and suppressing the Dll4-induced Notch signaling pathway in vivo. Also, H2 promotes the proliferation of HUVECs under hypoxia by negatively regulating the Dll4/Notch pathway and reducing ROS levels through Nrf2 pathway aligning with our findings in vivo.Moreover, the retinal oxygen-sensing mechanisms (HIF-1α/VEGF) are also involved in hydrogen-mediated retinal revascularization and neovascularization suppression. CONCLUSIONS: Collectively, our results indicate that H2 could be a promising therapeutic agent for POR treatment and that its beneficial effect in human ROP might involve the activation of the Nrf2-Notch axis as well as HIF-1α/VEGF pathways.

Impact of Saccharomyces boulardii CNCM I-745 on Bacterial Overgrowth and Composition of Intestinal Microbiota in Diarrhea-Predominant Irritable Bowel Syndrome Patients: Results of a Randomized Pilot Study.

BACKGROUND: Small intestinal bacterial overgrowth (SIBO) is associated with diarrhea-predominant irritable bowel syndrome (IBS-D). Probiotics like Saccharomyces boulardii CNCM I-745 (Sb) may be efficacious in balancing the microbiota. This randomized open label study assessed the effect of Sb in patients with bacterial overgrowth associated with IBS-D and its impact on the intestinal microbiota. METHODS: Patients were randomized to receive Sb + dietary advice (Sb + DA) or dietary advice (DA) only for 15 days. SIBO was assessed by the lactulose hydrogen breath test (LHBT). Symptoms were assessed with the IBS Symptom Severity Scale (IBS-SSS) and stool consistency with the Bristol Stool Form Scale. Microbiota and mycobiota were analyzed by 16S rDNA and ITS2. RESULTS: 54 patients were included, among whom 48 (27 Sb + DA, 21 DA) were evaluated. Decrease of hydrogen excretion was slightly higher in Sb + DA group, 41% versus 29% in DA group, and IBS-SSS total score were reduced by -134 and -93, respectively. The proportion of patients with diarrhea was lower in the Sb + DA group than in the DA group (25.9% compared to 47.6%). Bacterial and fungal microbiota showed that Sb treatment was associated with several modifications. Interestingly, F. prausnitzii was more abundant in Sb-treated patients with marked clinical improvement. The safety of S. boulardii CNCM I-745 was excellent. CONCLUSIONS: In patients with SIBO, S. boulardii CNCM I-745 associated with dietary advice reduced bacterial overgrowth and improved digestive symptoms while restoring the intestinal microbiota. The increased abundance of F. prausnitzii coupled with symptom improvement merits further research.

Bovine pancreatic trypsin inhibitor and soybean Kunitz trypsin inhibitor: Differential effects on proteases and larval development of the soybean pest Anticarsia gemmatalis (Lepidoptera: Noctuidae).

Pest management is challenged with resistant herbivores and problems regarding human health and environmental issues. Indeed, the greatest challenge to modern agriculture is to protect crops from pests and still maintain environmental quality. This study aimed to analyze by in silico, in vitro, and in vivo approaches to the feasibility of using the inhibitory protein extracted from mammals - Bovine Pancreatic Trypsin Inhibitor (BPTI) as a potential inhibitor of digestive trypsins from the pest Anticarsia gemmatalis and comparing the results with the host-plant inhibitor - Soybean Kunitz Trypsin Inhibitor (SKTI). BPTI and SKTI interacts with A. gemmatalis trypsin-like enzyme competitively, through hydrogen and hydrophobic bonds. A. gemmatalis larvae exposed to BPTI did not show two common adaptative mechanisms i.e., proteolytic degradation and overproduction of proteases, presenting highly reduced trypsin-like activity. On the other hand, SKTI-fed larvae did not show reduced trypsin-like activity, presenting overproduction of proteases and SKTI digestion. In addition, the larval survival was reduced by BPTI similarly to SKTI, and additionally caused a decrease in pupal weight. The non-plant protease inhibitor BPTI presents intriguing element to compose biopesticide formulations to help decrease the use of conventional refractory pesticides into integrated pest management programs.

Hydrogen-rich saline alleviates early brain injury through regulating of ER stress and autophagy after experimental subarachnoid hemorrhage.

PURPOSE: Subarachnoid hemorrhage (SAH) is a common complication of cerebral vascular disease. Hydrogen has been reported to alleviate early brain injury (EBI) through oxidative stress injury, reactive oxygen species (ROS), and autophagy. Autophagy is a programmed cell death mechanism that plays a vital role in neuronal cell death after SAH. However, the precise role of autophagy in hydrogen-mediated neuroprotection following SAH has not been confirmed. METHODS: In the present study, the objective was to investigate the neuroprotective effects and potential molecular mechanisms of hydrogen-rich saline in SAH-induced EBI by regulating neural autophagy in the C57BL/6 mice model. Mortality, neurological score, brain water content, ROS, malondialdehyde (MDA), and neuronal death were evaluated. RESULTS: The results show that hydrogen-rich saline treatment markedly increased the survival rate and neurological score, increased neuron survival, downregulated the autophagy protein expression of Beclin-1 and LC3, and endoplasmic reticulum (ER) stress. That indicates that hydrogen-rich saline-mediated inhibition of autophagy and ER stress ameliorate neuronal death after SAH. The neuroprotective capacity of hydrogen-rich saline is partly dependent on the ROS/Nrf2/heme oxygenase-1 (HO-1) signaling pathway. CONCLUSIONS: The results of this study demonstrate that hydrogen-rich saline improves neurological outcomes in mice and reduces neuronal death by protecting against neural autophagy and ER stress.

Hydrogen influences HDL-associated enzymes and reduces oxidized phospholipids levels in rats fed with a high-fat diet.

AIMS: Oxidized phospholipids (OxPLs) are formed as a result of oxidative stress, which potentially mediate multiple pathological effects. We aimed to evaluate the effects of hydrogen (H2) on OxPLs in vivo and the underlying mechanism. MAIN METHODS: Rats were randomly assigned to three groups: control group fed with a chow diet, model group fed with a high-fat diet, and H2-treated group fed with a high-fat diet and treated by 4% H2 inhalation for ten weeks. OxPLs in liver and plasma were analyzed by liquid chromatography-mass spectrometry. High-density lipoprotein (HDL) was separated by ultracentrifugation. A proteomic analysis was performed to reveal the alternation of HDL protein composition and he antioxidant capacity of HDL was tested by low-density lipoprotein oxidation experiment. Furthermore, the activity or expression of HDL-associated enzymes were evaluated. KEY FINDINGS: Inhalation of 4% H2 decreased the accumulation of OxPLs in rats. In vitro tests revealed that the different concentrations of H2 did not inhibit the formation of OxPLs mediated by non-enzymatic oxidation. H2 inhalation altered the components and enhanced the anti-oxidative capacity of HDL in rats fed with a high-fat diet. Further experiments showed that H2 significantly regulated the activity of lipoprotein-associated phospholipase A2, paraoxonase-1, and the expression of lecithin:cholesterol acyltransferase. SIGNIFICANCE: Our findings revealed that H2 may reduce the OxPLs levels through its influence on HDL-associated enzymes that can act on OxPLs, suggesting that H2 can be used in alleviating diseases related to lipid peroxidation due to oxidative stress.

Hydrogen-rich saline alleviates early brain injury through regulating of ER stress and autophagy after experimental subarachnoid hemorrhage

ABSTRACT Purpose: Subarachnoid hemorrhage (SAH) is a common complication of cerebral vascular disease. Hydrogen has been reported to alleviate early brain injury (EBI) through oxidative stress injury, reactive oxygen species (ROS), and autophagy. Autophagy is a programmed cell death mechanism that plays a vital role in neuronal cell death after SAH. However, the precise role of autophagy in hydrogen-mediated neuroprotection following SAH has not been confirmed. Methods: In the present study, the objective was to investigate the neuroprotective effects and potential molecular mechanisms of hydrogen-rich saline in SAH-induced EBI by regulating neural autophagy in the C57BL/6 mice model. Mortality, neurological score, brain water content, ROS, malondialdehyde (MDA), and neuronal death were evaluated. Results: The results show that hydrogen-rich saline treatment markedly increased the survival rate and neurological score, increased neuron survival, downregulated the autophagy protein expression of Beclin-1 and LC3, and endoplasmic reticulum (ER) stress. That indicates that hydrogen-rich saline-mediated inhibition of autophagy and ER stress ameliorate neuronal death after SAH. The neuroprotective capacity of hydrogen-rich saline is partly dependent on the ROS/Nrf2/heme oxygenase-1 (HO-1) signaling pathway. Conclusions: The results of this study demonstrate that hydrogen-rich saline improves neurological outcomes in mice and reduces neuronal death by protecting against neural autophagy and ER stress.

Chronic molecular hydrogen inhalation mitigates short and long-term memory loss in polymicrobial sepsis.

The central nervous system (CNS) is one of the first physiological systems to be affected in sepsis. During the exacerbated systemic inflammatory response at the early stage of sepsis, circulatory inflammatory mediators are able to reach the CNS leading to neuroinflammation and, consequently, long-term impairment in learning and memory formation is observed. The acute treatment with molecular hydrogen (H2) exerts important antioxidative, antiapoptotic, and anti-inflammatory effects in sepsis, but little is known about the mechanism itself and the efficacy of chronic H2 inhalation in sepsis treatment. Thus, we tested two hypotheses. We first hypothesized that chronic H2 inhalation is also an effective therapy to treat memory impairment induced by sepsis. The second hypothesis is that H2 treatment decreases sepsis-induced neuroinflammation in the hippocampus and prefrontal cortex, important areas related to short and long-term memory processing. Our results indicate that (1) chronic exposure of hydrogen gas is a simple, safe and promising therapeutic strategy to prevent memory loss in patients with sepsis and (2) acute H2 inhalation decreases neuroinflammation in memory-related areas and increases total nuclear factor E2-related factor 2 (Nrf2), a transcription factorthat regulates a vast group of antioxidant and inflammatory agents expression in these areas of septic animals.

Molecular hydrogen reduces acute exercise-induced inflammatory and oxidative stress status.

Physical exercise induces inflammatory and oxidative markers production in the skeletal muscle and this process is under the control of both endogenous and exogenous modulators. Recently, molecular hydrogen (H2) has been described as a therapeutic gas able to reduced oxidative stress in a number of conditions. However, nothing is known about its putative role in the inflammatory and oxidative status during a session of acute physical exercise in sedentary rats. Therefore, we tested the hypothesis that H2 attenuates both inflammation and oxidative stress induced by acute physical exercise. Rats ran at 80% of their maximum running velocity on a closed treadmill inhaling either the H2 gas (2% H2, 21% O2, balanced with N2) or the control gas (0% H2, 21% O2, balanced with N2) and were euthanized immediately or 3 h after exercise. We assessed plasma levels of inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6] and oxidative markers [superoxide dismutase (SOD), thiobarbituric acid reactive species (TBARS) and nitrite/nitrate (NOx)]. In addition, we evaluated the phosphorylation status of intracellular signaling proteins [glycogen synthase kinase type 3 (GSK3α/ß) and the cAMP responsive element binding protein (CREB)] that modulate several processes in the skeletal muscle during exercise, including changes in exercise-induced reactive oxygen species (ROS) production. As expected, physical exercise increased virtually all the analyzed parameters. In the running rats, H2 blunted exercise-induced plasma inflammatory cytokines (TNF-α and IL-6) surges. Regarding the oxidative stress markers, H2 caused further increases in exercise-induced SOD activity and attenuated the exercise-induced increases in TBARS 3 h after exercise. Moreover, GSK3α/ß phosphorylation was not affected by exercise or H2 inhalation. Otherwise, exercise caused an increased CREB phosphorylation which was attenuated by H2. These data are consistent with the notion that H2 plays a key role in decreasing exercise-induced inflammation, oxidative stress, and cellular stress.

Protective effect and mechanism of hydrogen treatment on lung epithelial barrier dysfunction in rats with sepsis.

This study aimed to explore the protective effect of hydrogen and to investigate the underlying mechanism of its preliminary effect on the alveolar epithelial barrier function in septic rats. Forty-five male Sprague-Dawley rats were divided randomly into three groups (N = 15): control [saline injection (intraperitoneal, ip), air drawing; SA], acute lung injury group [lipopolysaccharide (LPS) injection (ip, 15 mg/kg), air drawing; LA], and acute lung injury combined with hydrogen drawing group [LPS injection (ip, 15 mg/kg), 2% hydrogen drawing; LH]. The rats were euthanized after 6 h of treatment, and the extravascular lung water (EVLW), pulmonary alveolar-arterial oxygen pressure (A-aDO2), and respiratory index (RI) of each group were measured. The aquaporin-1 (AQP-1) protein expression in the lung tissues was detected using immunohistochemistry and western blotting, and the correlation between the EVLW and AQP-1 was analyzed. The lung morphology was observed with light and electron microscopy. In the LA group, EVLW (0.87 ± 0.17), A-aDO2 (113.21 ± 13.92), RI (0.65 ± 0.26), and AQP-1 expression increased. Additionally, thickened alveolar walls, significant invasion of inflammatory cells around the vessels, capillary ectasia, hyperemia/hemorrhage in the alveolar space, significantly swollen mitochondria, and increased vacuolar degeneration were observed. A significant negative correlation between AQP-1 expression and EVLW was observed (R2 = 0.8806). Compared with the LA group, EVLW (0.71 ± 0.19), A-aDO2 (132.42 ± 17.39), RI (0.75 ± 0.24), and inflammatory reaction decreased and AQP-1 expression increased in the LH group. The damage to pulmonary epithelial cells improved after hydrogen treatment in rats with sepsis; hydrogen could protect the pulmonary epithelial barrier function by acting on AQP-1.

Metformin induces apoptosis and cell cycle arrest mediated by oxidative stress, AMPK and FOXO3a in MCF-7 breast cancer cells.

Recent studies have demonstrated that the anti-diabetic drug, metformin, can exhibit direct antitumoral effects, or can indirectly decrease tumor proliferation by improving insulin sensitivity. Despite these recent advances, the underlying molecular mechanisms involved in decreasing tumor formation are not well understood. In this study, we examined the antiproliferative role and mechanism of action of metformin in MCF-7 cancer cells treated with 10 mM of metformin for 24, 48, and 72 hours. Using BrdU and the MTT assay, it was found that metformin demonstrated an antiproliferative effect in MCF-7 cells that occurred in a time- and concentration-dependent manner. Flow cytometry was used to analyze markers of cell cycle, apoptosis, necrosis and oxidative stress. Exposure to metformin induced cell cycle arrest in G0-G1 phase and increased cell apoptosis and necrosis, which were associated with increased oxidative stress. Gene and protein expression were determined in MCF-7 cells by real time RT-PCR and western blotting, respectively. In MCF-7 cells metformin decreased the activation of IRß, Akt and ERK1/2, increased p-AMPK, FOXO3a, p27, Bax and cleaved caspase-3, and decreased phosphorylation of p70S6K and Bcl-2 protein expression. Co-treatment with metformin and H2O2 increased oxidative stress which was associated with reduced cell number. In the presence of metformin, treating with SOD and catalase improved cell viability. Treatment with metformin resulted in an increase in p-p38 MAPK, catalase, MnSOD and Cu/Zn SOD protein expression. These results show that metformin has an antiproliferative effect associated with cell cycle arrest and apoptosis, which is mediated by oxidative stress, as well as AMPK and FOXO3a activation. Our study further reinforces the potential benefit of metformin in cancer treatment and provides novel mechanistic insight into its antiproliferative role.

Evaluation of the mean energy deposit during the impact of charged particles on liquid water.

The DNA strand break yield due to the impact of ionizing particles on living beings is closely related to the number of inelastic events per unit absorbed dose produced by these particles. The higher this number, the higher the probability of causing DNA strand breaks per unit absorbed dose. In a previous work, it was found that the total number of events produced by primary particles and the secondary electrons is almost independent of the type and energy of the incident particle (or LET). This finding could be supported by a quasi-constant mean energy deposit by inelastic event (ε). In this work, ε was defined and determined for electrons and the non-negative charge states of hydrogen (H°,⁺) and helium (He°,⁺,²âº) species impacting on liquid water. Ionization, excitation and charge transfer (up to two-electron transfers) processes have been included in present calculations. We found that, for liquid water, ε is within 13.7 ± 4.1 eV, 14.2 ± 1.7 eV and 13.8 ± 1.4 eV for electrons, hydrogen and helium species, respectively, with impact energies changing over three orders of magnitude. Unlike the mean excitation energy, the mean energy deposit per inelastic event depends not only on the target molecule but also on the projectile features. However, this dependence is relatively weak. This fact supports the quasi-independent number of inelastic events per unit absorbed dose found previously when charged particles impact on matter.

The hypothesis of an effective safe and novel radioprotective agent hydrogen-rich solution / La hipótesis de una solución rica en hidrógeno como agente radioprotector novedoso, seguro y efectivo

Most ionizing radiation-induced damage is caused by radical oxygen species (ROS). Some radioprotectors, such as amifostine, exert radioprotective effects by scavenging radical oxygen species. Recent studies show that hydrogen (H2) has antioxidant activities that protect the brain and intestine against ischaemia-reperfusion injury and stroke by selectively reducing hydroxyl and peroxynitrite radicals. However, it is seldom regarded as a radioprotective agent. In like manner, we hypothesize that hydrogen may be an effective, specific and novel radioprotective agent. But H2 is explosive, while hydrogen-rich solution (solution such as physiological saline saturated with molecular hydrogen) is safer.

La mayor parte de los efectos dañinos inducidos por la radiación ionizante, son causados por especies radicales de oxígeno (ROS). Algunos radioprotectores, tales como la amifostina, ejercen efectos radioprotectores mediante el rescate de especies radicales de oxígeno. Estudios recientes muestran que el hidrógeno (H2) posee una actividad antioxidante que protege el cerebro y el intestino contra las lesiones por repercusión isquémica y accidente cerebrovascular, mediante la reducción selectiva de radicales de hidroxilo y peroxinitrito. Sin embargo, raramente se le considera como un agente radioprotector. De manera similar, planteamos la hipótesis de que el hidrógeno puede ser un agente radioprotector efectivo, específico y novedoso. Pero el H2 es explosivo, mientras que la solución rica en hidrógeno (como es el caso del suero fisiológico saturado con hidrógeno molecular) es más segura.

High CO2/H+ dialysis in the caudal ventrolateral medulla (Loeschcke's area) increases ventilation in wakefulness.

Central chemoreception, the detection of CO(2)/H(+) within the brain and the resultant effect on ventilation, was initially localized at two areas on the ventrolateral medulla, one rostral (rVLM-Mitchell's) the other caudal (cVLM-Loeschcke's), by surface application of acidic solutions in anesthetized animals. Focal dialysis of a high CO(2)/H(+) artificial cerebrospinal fluid (aCSF) that produced a milder local pH change in unanesthetized rats (like that with a approximately 6.6mm Hg increase in arterial P(CO2)) delineated putative chemoreceptor regions for the rVLM at the retrotrapezoid nucleus and the rostral medullary raphe that function predominantly in wakefulness and sleep, respectively. Here we ask if chemoreception in the cVLM can be detected by mild focal stimulation and if it functions in a state dependent manner. At responsive sites just beneath Loeschcke's area, ventilation was increased by, on average, 17% (P<0.01) only in wakefulness. These data support our hypothesis that central chemoreception is a distributed property with some sites functioning in a state dependent manner.

The hypothesis of an effective safe and novel radioprotective agent: hydrogen-rich solution.

Most ionizing radiation-induced damage is caused by radical oxygen species (ROS). Some radioprotectors, such as amifostine, exert radioprotective effects by scavenging radical oxygen species. Recent studies show that hydrogen (H) has antioxidant activities that protect the brain and intestine against ischaemia-reperfusion injury and stroke by selectively reducing hydroxyl and peroxynitrite radicals. However it is seldom regarded as a radioprotective agent. In like manner we hypothesize that hydrogen may be an effective, specific and novel radioprotective agent. But H2 is explosive, while hydrogen-rich solution (solution such as physiological saline saturated with molecular hydrogen) is safer.

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance.

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80 degrees C) and under high pressure conditions at low temperature (3.75 kbar, -13 degrees C). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80°C) and under high pressure conditions at low temperature (3.75 kbar, -13°C). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

[Evaluation of antimicrobial drugs and atmospheres for the isolation of Campylobacter fetus subspp. from the bovine genital tract]. / Evaluación de drogas antimicrobianas y atmósferas para el aislamiento de Campylobacter fetus subspp. del tracto genital bovino.

Growth of 3 reference bovine C. fetus strains in media with and without antibiotics and bacteriostats active against the most common contaminant bacteria in the bovine genital tract was evaluated. In addition, 2 regional bovine C. fetus strains and 1 reference C. sputorum biovar bubulus strain were used in some experiments. Reference strain C. fetus subsp. venerealis was completely inhibited by polymyxin (> or = 0.25 IU/ml) whereas the other C. fetus strains were not inhibited. In Shepler's medium supplemented with rifampicin (10 micrograms/ml) subsp. fetus, was the only one to grow. When rifampicin was used at 5 micrograms/ml together with a reduced dose of the other Shepler's antibiotics, especially polymyxin B (0.85 IU/ml), subsp. venerealis was able to grow; nevertheless even at such a reduced dose, rifampicin was inhibitory for the biotype intermedius. It was demonstrated that triclosan (Irgasan) could be very useful at < or = 10 micrograms/ml in media with added blood, < or = 6 micrograms/ml in brucella broth and < or = 3 micrograms/ml in Mueller-Hinton broth for isolation of all subspecies of C. fetus. The sensitivity of C. fetus to 5-fluorouracil was variable: subsp. fetus was resistant (up to 800 micrograms/ml) whereas subspp. venerealis and biotype intermedius grew slowly or sometimes did not grow at all in concentrations of 6.25 micrograms/ml onwards. Fosfomycin was inhibitory to all C. fetus strains at > or = 50 micrograms/ml. C. sputorum biovar. bubulus was less inhibited than C. fetus with triclosan grew up to more than 100 micrograms/ml, with 5-fluorouracil up to 100 micrograms/ml and with fosfomycin up to 50 micrograms/ml. Growth of C. fetus subspp. was compared in different microaerophilic atmospheres contained in anaerobic jars (Oxoid HP 11) without palladium catalyzer. Growth with nitrogen or hydrogen was similar. When jars were replaced by 15 x 13 cm cylindrical cans without valves or gas measurement devices only pure hydrogen supported satisfactory growth of all C. fetus subspp. and C. sputorum biovar. bubulus strains. The candle system, a commercial nitrogen rich gas mixture and pure carbonic anhydride prepared in these cans failed to enable these strains to grow. C. fetus subsp. fetus was more aero-tolerant than subsp. venerealis and its biotype intermedius and was able to grow, although very weakly, with only carbonic anhydride added to an aerobic atmosphere. The growth obtained using a commercial gas generating microaerophilic kit (Oxoid BR-56) was comparable to the one achieved with the hydrogen rich atmosphere prepared in our laboratory.