Time-resolved fluoroimmunoassay of potato virus M with monoclonal antibodies

Mouse monoclonal antibodies (MAbs) specific for potato virus M (PVM) were prepared and the properties of three of them were studied. MAb M4C1 is IgG2b, it binds with high affinity to PVM coat protein, to purified virus preparations and recognises PVM in infected potato leaves and tubers. MAb M6D5 is IgG2a and also reacts with PVM coat protein, purified PVM and with PVM in potato leaf and tuber extracts. In double-antibody sandwich ELISA (DAS ELISA) MAbs M4C1 and M6D5 reacted with all 17 PVM isolates tested. MAb M7 is IgG2b and recognises PVM only in indirect dot ELISA on nitrocellulose filters and viral coat protein on Western blots. MAbs against PVM were used as capture antibodies and europium-labelled MAbs as conjugates in time-resolved fluoroimmunoassay (EuTRFIA). The standard EuTRFIA curve of PVM detection is approximately linear over a range of PVM concentrations from 0.5 ng/ml to 1000 ng/ml. The lowest PVM concentration detectable in EuTRFIA was 0.5 ng/ml and correspondingly 6 ng/ml in DAS ELISA. The use of the europium chelate label allows PVM detection in potato leaf and tuber sap at dilutions greater than 10^--⁴ with very low background fluorescence. EuTRFIA with MAbs, with either one or two incubations is about 10–20 times more sensitive for PVM detection than is DAS ELISA. PVM and PVX, mixed with healthy potato tuber sap, were simultaneously tested in a single sample at concentrations lower than 10 ng/ml by double-label TRFIA using europium-labelled MAbs to PVM and samarium-labelled MAbs to PVX.     

Mineral content of leaves from trees growing on serpentine soils under contrasting rainfall regimes in Puerto Rico

The interactions between water and soil nutrient availability in determining leaf nutritional composition and structural features were investigated in forests on serpentine in Maricao and Susua (Puerto Rico). These forests grow under contrasting rainfall regimes: Maricao is a wet forest located at altitudes above 500 m and receiving more than 2500 mm rainfall, while Susua is a humid forest located well below 500 m, with less than 1500 mm rainfall and a well defined dry season. Dominant tree species and soils were analysed for N, P, K, Ca, Mg and Ni. Soils can be differentiated according to their K content (higher in Maricao) and P contents (higher in Susua). Mature leaves of both forests have sclerophyllous characteristics as judged from the Specific Leaf Areas (<80 cm² g^-1) and low P contents. Leaf area development is strongly correlated with leaf N and P contents in both forests, but Maricao samples appear to be more limited by P availability. In concordance with soil values, the Susua leaf sample set has significantly higher contents of P, but lower contents of K when compared with the Maricao sample set. Analyses of soluble K, Ca, and Mg reveal strong physiological selectivity in the absorption of these cations. K/Ca and Ca/Mg ratios are markedly higher in the soluble leaf extracts than in the soil extracts. It seems that restriction to vegetation development in the serpentine areas investigated are more related to nutritional deficiencies and not to high contents of either Mg or Ni in the upper soil layers. Only two strong Ni accumulators were found, Cassine xylocarpa (1.2 mol Ni g^-1 dry mass or 70 g g^-1) from Susua, and Chionanthus domingensis (12.2 mol g^-1, or about 700 g g^-1) from Maricao. These species are not restricted to serpentine areas in Puerto Rico.     

Isolation of thymidine kinase-deficient rat hepatoma cells by selection with bromodeoxyuridine,Hoechst 33258, and visible light

The photosensitivity of bromodeoxyuridine (BrdU)-substituted cells is known to be markedly enhanced by the fluorochrome Hoechst 33258. Since the incorporation of BrdU into nucleic acids depends upon its prior phosphorylation via thymidine kinase (TK; EC 2.7.1.21), cells deficient in TK activity are refractory to photoinduced killing. These observations suggested that combined treatment with BrdU, Hoechst 33258, and visible light would constitute an efficient selective strategy for the recovery of TK^– mutant cells. In this report we describe a single-step selection protocol which reduced the survival of TK⁺ cells by a factor of 10⁵ without affecting the viability of TK^– mutants. This procedure was used to isolate H4IIEC3-derived rat hepatoma cells deficient in TK activity. The properties of several TK^– hepatoma clones are discussed.The valuable technical assistance of J. M. Courvall, F. R. Parker, and M. M. Smith is acknowledged. These studies were supported by grant GM26449 from the National Institutes of Health. A.M.K. was supported by a postdoctoral fellowship from the American Cancer Society, California Division. T.G.L. is a Leukemia Society of America postdoctoral fellow. R.E.K.F. is the recipient of an American Cancer Society Faculty Research Award.     

Antimicrobial and antioxidant activities of Mexican oregano essential oils (Lippia graveolens H. B. K.) with different composition when microencapsulated inβ‐cyclodextrin

Aims: To study how the antimicrobial and antioxidant activities of Lippia graveolens essential oils with different composition are affected after the microencapsulation process with β‐cyclodextrin (βCD). Methods and results: Three Mexican oregano essential oils (EOs) with different carvacrol/thymol/p‐cymene ratios (38 : 3 : 32, 23 : 2 : 42, 7 : 19 : 35) were used in this study. Microencapsulation was carried out by spray‐drying. Antimicrobial activities were measured as MBC (minimal bactericidal concentration) using 0·05%/0·10%/0·20% (w/v) dilutions of EOs against Escherichia coli ATCC 11229, Pseudomonas aeruginosa ATCC 9027 and Staphylococcus aureus ATCC 6538. Antioxidant activities were determined by the 2,2′‐diphenyl‐1‐picrylhydrazil (DPPH) method. EOs showed antimicrobial and antioxidant activity, but microencapsulation preserved the antimicrobial activity in all cases and increased the antioxidant activity from four‐ to eightfold. Conclusions: Although the Lippia essential oils were from the same species, their composition affects the biological activities before and after the microencapsulation process, as well as encapsulation efficiency. Our study supports the fact that microencapsulation of EOs in β‐cyclodextrin preserves the antimicrobial activity, improves the antioxidant activity and acts as a protection for EOs main compounds. Significance and Impact of the Study: Microencapsulation affects positively EOs main compounds, improves antioxidant activity and retains antimicrobial activity, enhancing the quality of the oils.     

Structural and thermodynamic folding characterization of triosephosphate isomerases from Trichomonas vaginalis reveals the role of destabilizing mutations following gene duplication

We report the structures and thermodynamic analysis of the unfolding of two triosephosphate isomerases (TvTIM1 and TvTIM2) from Trichomonas vaginalis. Both isoforms differ by the character of four amino acids: E/Q 18, I/V 24, I/V 45, and P/A 239. Despite the high sequence and structural similarities between both isoforms, they display substantial differences in their stabilities. TvTIM1 (E18, I24, I45, and P239) is more stable and less dissociable than TvTIM2 (Q18, V24, V45, and A239). We postulate that the identities of residues 24 and 45 are responsible for the differences in monomer stability and dimer dissociability, respectively. The structural difference between both amino acids is one methyl group. In TvTIMs, residue 24 is involved in packing α‐helix 1 against α‐helix 2 of each monomer and residue 45 is located at the center of the dimer interface forming a “ball and socket” interplay with a hydrophobic cavity. The mutation of valine at position 45 for an alanine in TvTIM2 produces a protein that migrates as a monomer by gel filtration. A comparison with known TIM structures indicates that this kind of interplay is a conserved feature that stabilizes dimeric TIM structures. In addition, TvTIMs are located in the cytoplasm and in the membrane. As TvTIM2 is an easily dissociable dimer, the dual localization of TvTIMs may be related to the acquisition of a moonlighting activity of monomeric TvTIM2. To our knowledge, this is the simplest example of how a single amino acid substitution can provide alternative function to a TIM barrel protein. Proteins 2014; 82:22–33. © 2013 Wiley Periodicals, Inc.     

Changes in Structure,Composition, and Nutrients During 15 Yr of Hurricane-Induced Succession in a Subtropical Wet Forest in Puerto Rico

The trajectory of hurricane-induced succession was evaluated in a network of forest plots measured immediately before and 3 mo, 5, 10, and 15 yr after the direct impact of a Category 4 hurricane. Comparisons of forest structure, composition, and aboveground nutrients pools were made through time, and between species, life-history groups and geomorphic settings. The hurricane reduced aboveground biomass by 50 percent, causing an immediate decrease in stem density and diversity indices among all geomorphic settings. After 15 yr, basal area and aboveground biomass returned to pre-hurricane levels, while species richness, diversity indices, and stem densities exceeded pre-hurricane levels. Differences in species composition among geomorphic settings had not returned after 15 yr but differences in stem densities and structure were beginning to emerge. Significant differences were observed in the nutrient concentration of the three species that comprised the most aboveground biomass, and between species categorized as secondary high-light species and primary, low-light species. Species whose abundance was negatively correlated with the mature forest dominant also had distinct nutrient concentrations. When total aboveground nutrient pools were compared over time, differences in leaf nutrients among species were hidden by similarities in wood nutrient concentrations and the biomass dominance of a few species. The observed successional trajectory indicates that changes in species composition contributed to fast recovery of aboveground biomass and nutrient pools, while the influence of geomorphic setting on species composition occurs at time scales >15 yr of succession.     

Let's Not Forget the Biodiversity of the Cities

Cities contain over half of the world's population and are the source of many anthropogenic effects on the world's biota. Close examination of San Juan, Puerto Rico, a tropical city, reveals a significant green infrastructure. The city contains natural and human-constructed forests, urban aquatic systems with native and introduced species, and a thriving and diverse biota adapted to urban conditions. It behooves tropical ecologists to pay attention to the biodiversity of urban environments to learn lessons about ecological persistence and adaptation to novel anthropogenic environments.