Effect of freezing and storage temperature on stability and antimicrobial activity of an antibiotic mixture used for decontamination of tissue allografts

One of the most important risks to be controlled in tissue banking is the infection associated with the clinical use of auto- and allografts. Thus, tissue disinfection protocols are used, in addition to processing in controlled environments. For this purpose, combinations of antibiotics are designed to ensure a broad spectrum of antimicrobial activity. This type of protocol is usually validated by testing its antimicrobial efficacy. In this work, we have studied the effect of several factors on the potential of an antibiotic mixture: container, freezing, storage at 4 °C, storage at ??30 °C and storage at ??80 °C. The molecular stability of the compounds has also been tested, additionally to their efficacy. Our findings show that storage conditions affect the molecular stability of Fungizone and Tobramycin (only in case of frozen storage for the last one). Nevertheless, the solution retains its antimicrobial activity for several weeks. The availability of stored aliquots of disinfectant solution and defining expiry dates for different storage conditions can help to schedule tissue bank tasks.     

Seed predation of two alien Opuntia species invading Mediterranean communities

Invasibility depends on the interaction of the introduced species with the abiotic and biotic factors of the recipient community. In particular, the biotic resistance posed by native herbivores has been claimed to be of great importance in controlling plant invasion. We investigated fruit and seed predation of two exotic Opuntia species within and between Mediterranean communities in order to determine how patterns of predation matched patterns of invasion. Predators were small mammals, presumably mice, which could consume more than 50% of the seeds produced. Predators could be equally effective in consuming fruit and single seeds. O. maxima fruits were slightly preferred to O. stricta fruits, but predators did not distinguish between seeds. Seed predation was more intense in invaded than in non-invaded communities. However, there was a high spatial variation in seed predation that did not always match patterns of invasion, suggesting that seed predation alone is not a good predictor of community invasibility to Opuntia. According to these results invasibility to Opuntia is limited in some (but not all) communities by native mice. Seed losses by predation were high for both species. However, we estimated that more than 75% of seeds dispersed by birds to non-invaded areas are not predated.     

The physiological and nutritional responses to an excess of boron by Verna lemon trees that were grafted on four contrasting rootstocks

Citrus species are sensitive to an excess of boron (B). Currently, this toxicity is becoming a serious problem in the soils of arid and semi-arid environments throughout the world, where high concentrations of B may occur due to the agricultural use of wastewater. Citrus rootstocks can greatly influence the tolerance of citrus trees to different abiotic stresses. However, little is known about how the rootstock influences the tolerance of these trees to an excess of B. In this study, the effects of the nutrient solution’s B concentration (0.25, 2, 4.5 or 7?mg?l^?1) on the growth and other physiological, nutritional and biochemical parameters of Verna lemon trees that were grafted on four contrasting rootstocks [Carrizo citrange (CC), Cleopatra mandarin (CL), Citrus macrophylla (CM) and sour orange (SO)] were investigated. The plants were grown in a greenhouse in pots containing a universal substrate media and were watered daily with a Hoagland nutrient solution containing different concentrations of B. The results showed that the plant growth was progressively inhibited with an increasing concentration of B in the nutrient solution. However, the shoot was more sensitive to the B toxicity than were the roots. In addition, the growth inhibition was reduced in trees that were grafted on CL and CM when compared with those that were grafted on CC and SO. The concentration of B in the leaves, stems and roots also increased with an increase in the concentration of external B in the following order: leaves?>?roots?>?stem. The rootstock also had an influence on the B concentration in the different plant tissues. In the leaves, the B concentration was lowest in the plants that were grafted on the SO rootstock followed by the plants that were grafted on either the CM or CL rootstock and highest in the plants that were grafted on the CC rootstock. The net assimilation of CO₂ ( $ A_{{{\text{CO}}_{2} }} $ ) and the stomatal conductance (g _s) leaf gas exchange parameters were reduced with an excess of B in the leaves, and this reduction was less pronounced for trees on CM and CL. The intercellular CO₂ concentration (C _i) and the chlorophyll fluorescence indicated that the reduction of $ A_{{{\text{CO}}_{2} }} $ that was found with an excess of B was mainly due to non-stomatal factors. The mineral nutrition and organic solute data are also shown in this study. All of the data indicate that the tolerance to an excess of B is not related to the concentration of B that has accumulated in the leaves, which indicates that a combination of rootstock-dependent physiological, biochemical and anatomical responses determine the tolerance to an excess of B in citrus plants.     

The Depletion of Nuclear Glutathione Impairs Cell Proliferation in 3t3 Fibroblasts

Background

Glutathione is considered essential for survival in mammalian cells and yeast but not in prokaryotic cells. The presence of a nuclear pool of glutathione has been demonstrated but its role in cellular proliferation and differentiation is still a matter of debate.

Principal Findings

We have studied proliferation of 3T3 fibroblasts for a period of 5 days. Cells were treated with two well known depleting agents, diethyl maleate (DEM) and buthionine sulfoximine (BSO), and the cellular and nuclear glutathione levels were assessed by analytical and confocal microscopic techniques, respectively. Both agents decreased total cellular glutathione although depletion by BSO was more sustained. However, the nuclear glutathione pool resisted depletion by BSO but not with DEM. Interestingly, cell proliferation was impaired by DEM, but not by BSO. Treating the cells simultaneously with DEM and with glutathione ethyl ester to restore intracellular GSH levels completely prevented the effects of DEM on cell proliferation.

Conclusions

Our results demonstrate the importance of nuclear glutathione in the control of cell proliferation in 3T3 fibroblasts and suggest that a reduced nuclear environment is necessary for cells to progress in the cell cycle.     

Role of endogenous and exogenous prostaglandins on the contractile functioning of isolated sow (Sus scrofa) oviducts

The output prostaglandins (PHs) E₁, E₂ and F₂ α, from ampullary and isthmic portions of sow oviducts isolated during proestrus, estrus and metestrus, was explored. Moreover, $\text{in vitro}$ cumulative dose-response curves for the contractile effect of these three PGs, on identical oviductal segments, were constructed. Isthmic preparations form proestrous and metestrous animals released more PGE₁ and PGF₂ α than PGE₂ “like material”. During estrus, the outputs of PGE₁, PGE₂ and PGF₂ α were similar, whereas, oviducts from proestrous and metestrous sows released less PGE₁ and PGF₂ α than during estrus. Although the output of PGE₂ “like material” from isthmic and ampullary segments did not differ significantly during the three stages of the sex cycle, ampullary metestrous preparations released more PGE₁ and PGF₂ α, than estrous or proestrous ones. The addition of PGE₁, PGE₂ α, consistently stimulatedthe amplitude of contractions of isthmic oviductal segments isolated from proestrous and metestrous sows. Within the concentration-range explored, dose-response curves for PGE₂ and PGE₁ were to the left of those for PGF₂ α in the isthmus obtained before ovulation (proestrus) but not in segments isolated at later times (2–3 days) of the cycle (metestrus). The stimulatory dose-response curves for PGE₁, or PGE₂, in isthmic segments of metestrous preparations incubated with phentolamine (10^?6M) were shifted to the right of controls not exposed to the adrenoreceptor blocker, whereas, the curve for PGF₂ α without phentolamine, was identical to that obtained in its presence. PGE₁ and PGE₂ did not evoke significant contractile effects on oviductal ampullary protions from proestrous sows, wherea, PGF₂ α was clearly stimulatory at concentrations of 10^?9M and higher. In ampullary segments isolated after ovulation (metestrus) the threshold for contractile enhancement following PGF₂ α was greater than during proestrus, whereas, PGE₁ elicited a significant inhibition of contractions. The spontaneous contractile pattern exhibited by isthmic and ampullary oviductal regions, prior to and after ovulation, is discussed in terms of tissue PG generation and output and is compared with results regarding tubal motility following the exposure to exogenous PGs.     

Prostacyclin induces a surprising long-lasting motility in quiescent uterine strips (indomethacin-treated) isolated from ovariectomized rats

Dose-response curves for several prostaglandins (PGI₂; PGD₂; PGF₂ and PGE₂); BaCl₂ or prostaglandin metabolites (15-keto-PGF_2α; 13, 14-diOH-15-keto-PGF_2α; 6-keto-PGF_1α and 6-keto-PGE₁ in quiescent (indomethacin-treated) uterine strips from ovariectomized rats, were constructed. All PGs tested as well as BaCl₂, triggered at different concentrations, evident phasic contractions. Within the range of concentrations tested the portion of the curves for the metabolites of PGF_2α was shifted to the right of that for PGF_2α itself; the curve for 6-keto-PGF_1α was displaced to the right of the curve for PGI₂ and that for 6-keto-PGE₁ to the left.It was also demonstrated that the uterine motility elicited by 10⁻⁵ M PGF_2α and its metabolites was long lasting (more than 3 hours) and so it was the activity evoked by PGI₂; 6-keto-PGF_1α and BaCl₂, but not the contractions following 6-keto-PGE₁, which disappeared much earlier. The contractile tension after PGF_2α; 15-keto-PGF_2α; 13, 14-diOH-15-keto-PGF_2α and PGI₂, increased as time progressed whilst that evoked by 6-keto-PGF_2α or BaCl₂ fluctuated during the same period around more constant levels.The surprising sustained and gradually increasing contractile activity after a single dose of an unstable prostaglandin such as PGI₂, on the isolated rat uterus rendered quiescent by indomethacin, is discussed in terms of an effect associated to its transformation into more stable metabolites (6-keto-PGF_1α, or another not tested) or as a consequence of a factor which might protects prostacyclin from inactivation.     

Inotropic effect of PGE1 and PGE2 on isolated rat atria. Influence of adrenergic mechanisms

The effects of a wide range of PGE1 and PGE2 concentrations on the isometric developed tension of isolated rat atria beating spontaneously or paced at a fixed rate, were explored. PGE1 only produced a negative inotropic effect (NIE), whereas PGE2 elicited a biphasic inotropic action; negative at low concentrations and positive (PIE) at higher ones. Phenoxybenzamine and phentolamine failed to modify either the NIE or the PIE, but subthreshold exogenous norepinephrine abolished the NIE, suggesting a presynaptic inhibitory effect of PGEs on the adrenergic neurotransmitter release. Auricles pretreated with subthreshold norepinephrine react with a PIE to PGE1, but not to PGE2. On the contrary in the presence of subthreshold methoxamine the PIE of PGE2 was increased whereas the action of PGE1 was not modified.     

SHR3: a novel component of the secretory pathway specifically required for localization of amino acid permeases in yeast.

Mutations in SHR3 block amino acid uptake into yeast by reducing the levels of multiple amino acid permeases within the plasma membrane. SHR3 is a novel integral membrane protein component of the endoplasmic reticulum (ER). shr3 null mutants specifically accumulate amino acid permeases in the ER; other plasma membrane proteins, secretory proteins, and vacuolar proteins are processed and targeted correctly. Our findings suggest that SHR3 interacts with a structural domain shared by amino acid permeases, an interaction required for permease-specific processing and transport from the ER. Even in the presence of excess amino acids, shr3 mutants exhibit starvation responses. shr3 mutants constitutively express elevated levels of GCN4, and mutant shr3/shr3 diploids undergo dimorphic transitions that result in filamentous growth at enhanced frequencies.     

Human platelet rich plasma and human serum protects from inactivation the antiaggregatory capacity of prostacyclin-like material (PGI2) produced by the rat stomach fundus

Prostacyclin (PGI₂) synthetizing capacity of rat stomach fundus in Krebs-Ringer-Bicarbonate (KRB); human platelet rich plasma (PRP) or human serum (HS), was explored. The basal production of PGI₂-like material was similar in the three media, suggesting the absence of any special substance in plasma or serum able to modify prostacyclin synthesis from tissue substrate. On the other hand it was also documented that in PRP and in HS the antiaggregatory activity of the PGI₂-like material declined in its capacity less than 50% following 60 minutes of incubation at 37 °C, whereas it almost dissapeared when incubated in KRB. Possible explanations underlying such finding are discussed.