Effect of immobilization support, water activity, and enzyme ionization state on cutinase activity and enantioselectivity in organic media

We studied the reaction between vinyl butyrate and 2-phenyl-1-propanol in acetonitrile catalyzed by Fusarium solani pisi cutinase immobilized on zeolites NaA and NaY and on Accurel PA-6. The choice of 2-phenyl-1-propanol was based on modeling studies that suggested moderate cutinase enantioselectivity towards this substrate. With all the supports, initial rates of transesterification were higher at a water activity (a(w)) of 0.2 than at a(w) = 0.7, and the reverse was true for initial rates of hydrolysis. By providing acid-base control in the medium through the use of solid-state buffers that control the parameter pH-pNa, which we monitored using an organo-soluble chromoionophoric indicator, we were able, in some cases, to completely eliminate dissolved butyric acid. However, none of the buffers used were able to improve the rates of transesterification relative to the blanks (no added buffer) when the enzyme was immobilized at an optimum pH of 8.5. When the enzyme was immobilized at pH 5 and exhibited only marginal activity, however, even a relatively acidic buffer with a pK(a) of 4.3 was able to restore catalytic activity to about 20% of that displayed for a pH of immobilization of 8.5, at otherwise identical conditions. As a(w) was increased from 0.2 to 0.7, rates of transesterification first increased slightly and then decreased. Rates of hydrolysis showed a steady increase in that a(w) range, and so did total initial reaction rates. The presence or absence of the buffers did not impact on the competition between transesterification and hydrolysis, regardless of whether the butyric acid formed remained as such in the reaction medium or was eliminated from the microenvironment of the enzyme through conversion into an insoluble salt. Cutinase enantioselectivity towards 2-phenyl-1-propanol was indeed low and was not affected by differences in immobilization support, enzyme protonation state, or a(w).     

The effects of l-DOPA on root growth, lignification and enzyme activity in soybean seedlings

In the present study, we investigated the effects of l-DOPA (l-3,4-dihydroxyphenylalanine), an allelochemical exuded from the velvetbean (Mucuna pruriens L DC. var. utilis), on the growth and cell viability of soybean (Glycine max L. Merrill) roots. We analyzed the effects of l-DOPA on phenylalanine ammonia lyase (PAL), cinnamyl-alcohol dehydrogenase (CAD) and cell wall-bound peroxidase (POD) activities as well as its effects on phenylalanine, tyrosine and lignin contents in the roots. 3-day-old seedlings were cultivated in half-strength Hoagland nutrient solution (pH 6.0), with or without 0.5?mM l-DOPA, in a growth chamber at 25?°C for 6, 12, 18 or 24?h with a day/night regime of 1:1, and a photon flux density of 280???mol?m^?2 s^?1. In general, the length, fresh weight and dry weight of the roots decreased followed by a significant loss of cell viability. Phenylalanine, tyrosine and lignin contents as well as PAL, CAD and cell wall-bound POD activities increased after l-DOPA treatment. These results reinforce the susceptibility of soybean to l-DOPA, which increases the enzyme activity in the phenylpropanoid pathway and, therefore, provides precursors for the polymerization of lignin. In brief, these findings suggest that the inhibition of soybean root growth induced by exogenously applied l-DOPA may be due to excessive production of lignin in the cell wall.     

Synthesis and induction of apoptosis signaling pathway of ent-kaurane derivatives

Thirty one ent-kaurane derivatives were prepared from kaurenoic acid (1), grandiflorenic acid (16), 15α-acetoxy-kaurenoic acid (26) and 16α-hydroxy-kaurenoic acid (31). They were tested for their ability to inhibit cell viability in the mouse leukemic macrophagic RAW 264.7 cell line. The most effective compounds were 12, 20, 21, and 23. These were selected for further evaluation in other human cancer cell lines such as Hela, HepG2, and HT-29. Similar effects were obtained although RAW 264.7 cells were more sensitive. In addition, these compounds were significantly less cytotoxic in non-transformed cells. The apoptotic potential of the most active compounds was investigated and they were able to induce apoptosis with compound 12 being the best inducer. The caspase-3, -8 and -9 activities were measured. The results obtained showed that compounds 12, 21, and 23 induce apoptosis via the activation of caspase-8, whereas compound 20 induces apoptosis via caspase-9. Immunoblot analysis of the expression of p53, Bax, Bcl-2, Bcl-xl, and IAPs in RAW 264.7 cells was also carried out. When cells were exposed to 5 μM of the different compounds, expression levels of p53 and Bax increased whereas levels of antiapoptotic proteins such as Bc1-2, Bc1-x1, and IAPs decreased. In conclusion, kaurane derivatives (12, 20, 21, and 23) induce apoptosis via both the mitochondrial and membrane death receptor pathways, involving the Bcl-2 family proteins. Taken together these results provide a role of kaurane derivatives as apoptotic inducers in tumor cells.     

Mitogen-activated protein kinases modulate H(2)O(2)-induced apoptosis in primary rat alveolar epithelial cells

Increasing evidence suggests a role for apoptosis in the maintenance of the alveolar epithelium under normal and pathological conditions. However, the signaling pathways modulating alveolar type II (ATII) cell apoptosis remain poorly defined. Here we investigated the role of MAPKs as modulators of oxidant-mediated ATII cell apoptosis using in vitro models of H(2)O(2)-stress. H(2)O(2), delivered either as a bolus or as a flux, lead to time- and concentration-dependent increases in ATII cells apoptosis. Increased apoptosis in primary rat ATII cells was detected at H(2)O(2) concentrations and production rates in the physiological range (1 microM) and peaked at 100 microM H(2)O(2). Immortalized rat lung epithelial cells (RLE), in contrast, required millimolar concentration of H(2)O(2) for maximal responses. H(2)O(2)-induced apoptosis was preceded by rapid activation of all three classes of mitogen-activated protein kinases (MAPKs): ERK, JNK, and p38. Specific inhibition of JNK using antisense oligonucleotides and ERK and p38 using PD98059 or SB202190, respectively, indicated a pro-apoptotic role for JNK pathway and an anti-apoptotic role for ERK- and p38-initiated signaling events. Our data show that the balance between the activation of JNK, ERK, and p38 is a critical determinant of cell fate, suggesting that pharmacological interventions on the MAPK pathways may be useful in the treatment of oxidant-related lung injury.     

Long-Lasting Immune Protection and Other Epidemiological Findings after Chikungunya Emergence in a Cambodian Rural Community,April 2012

The East/Central/South African genotype of Chikungunya virus with the E1-A226V mutation emerged in 2011 in Cambodia and spread in 2012. An outbreak of 190 cases was documented in Trapeang Roka, a rural village. We surveyed 425 village residents within 3–4 weeks after the outbreak, and determined the sensitivity and specificity of case definitions and factors associated with infection by CHIKV. Self-reported clinical presentation consisted mostly of fever, rash and arthralgia. The presence of all three clinical signs or symptoms was identified as the most sensitive (67%) and specific (84%) self-reported diagnostic clinical indicator compared to biological confirmation by MAC-ELISA or RT-PCR used as a reference. Having an indoor occupation was associated with lower odds of infection compared with people who remained at home (adjOR 0.32, 95%CI 0.12–0.82). In contrast with findings from outbreaks in other settings, persons aged above 40 years were less at risk of CHIKV infection, likely reflecting immune protection acquired when Chikungunya circulated in Cambodia before the Khmer Rouge regime in 1975. In view of the very particular history of Cambodia, our epidemiological data from Trapeang Roka are the first to support the persistence of CHIKV antibodies over a period of 40 years.     

Phylogenetic and molecular clock inferences of cyanobacterial strains within Rivulariaceae from distant environments

Heterocyst-forming cyanobacteria are important players at both evolutionary and ecological scales, but to date it has been difficult to establish their phylogenetic affiliations. We present data from a phylogenetic and molecular clock analysis of heterocystous cyanobacteria within the family Rivulariaceae, including the genera Calothrix, Rivularia, Gloeotrichia and Tolypothrix. The strains were isolated from distant geographic regions including fresh and brackish water bodies, microbial mats from beach rock, microbialites, pebble beaches, plus PCC strains 7103 and 7504. Phylogenetic inferences (distance, likelihood and Bayesian) suggested the monophyly of genera Calothrix and Rivularia. Molecular clock estimates indicate that Calothrix and Rivularia originated ～1500 million years ago (MYA) ago and species date back to 400-300 MYA while Tolypothrix and Gloeotrichia are younger genera (600-400 MYA).     

Calculation of NMR relaxation, covolume, and scattering-related properties of bead models using the SOLPRO computer program

The hydrodynamic properties of macromolecules and bioparticles, represented by bead models, can be calculated using methods implemented in the computer routine HYDRO. Recently, a new computer routine, SOLPRO, has been presented for the calculation of various SOLution PROperties. These include (1) time-dependent electro-optic and spectroscopic properties related to rotational diffusion, (2) non-dynamic properties like scattering curves, and (3) dimensionless quantities that combine two or more solution properties in a form which depends on the shape of the macromolecule but not on its size. In the present work we describe the inclusion of more of those types of properties in a new version of SOLPRO. Particularly, we describe the calculation of relaxation rates in nuclear magnetic resonance (NMR). For dipolar coupling, given the direction of the dipole the program calculates values of the spectral density, from which the NMR relaxation times can be obtained. We also consider scattering-related properties, namely the distribution of distances for the bead model, which is directly related to the angular dependence of scattered intensity, and the particle's longest distance. We have devised and programmed a procedure to calculate the covolume of the bead model, related to the second virial coefficient and, in general, to the concentration dependence of solution properties. Various shape-dependent dimensionless quantities involving the covolume are calculated. In this paper we also discuss some aspects, namely bead overlapping and hydration, that are not explicitely included in SOLPRO, but should be considered by the user. Received: 25 May 1998 / Revised version: 30 July 1998 / Accepted: 30 July 1998     

Anopheles albitarsis eggs: ultrastructural analysis of chorion layers after permeabilization

Construction of transgenic Anopheles mosquitoes refractory to Plasmodium requires knowledge of mosquito developmental biology. In order to study Anopheles embryology the removal or, alternatively, the permeabilization of the melanized and sclerotized egg chorion were attempted. The protocol classically used for chorion removal of Drosophila eggs was applied, with partial efficacy, to Anopheles albitarsis, a neotropical malaria vector. Each step was monitored by scanning electron microscopy and the results suggest differences in chorion composition between the two taxa. As an alternative to chorion removal, mosquito eggs were permeabilized with benserazide, an inhibitor of Dopa Decarboxylase, one of the enzymes needed for mosquito eggshell sclerotization. Embryo morphology and viability were not affected by this treatment. Permeabilization of the egg chorion allowed the ultrastructural observation of an internal homogeneous endochorion and an external compound exochorion, the latter consisting of a basal lamellar layer and protruding tubercles.