A rapid,ideal, and eco-friendlier protocol for quantifying proline

Proline, a stress marker, is routinely quantified by a protocol that essentially uses hazardous toluene. Negative impacts of toluene on human health prompted us to develop a reliable alternate protocol for proline quantification. Absorbance of the proline-ninhydrin condensation product formed by reaction of proline with ninhydrin at 100 °C in the reaction mixture was significantly higher than that recorded after its transfer to toluene, revealing that toluene lowers sensitivity of this assay. λ _max of the proline-ninhydrin complex in the reaction mixture and toluene were 508 and 513 nm, respectively. Ninhydrin in glacial acetic acid yielded higher quantity of the proline-ninhydrin condensation product compared to ninhydrin in mixture of glacial acetic acid and H₃PO₄, indicating negative impact of H₃PO₄ on proline quantification. Further, maximum yield of the proline-ninhydrin complex with ninhydrin in glacial acetic acid and ninhydrin in mixture of glacial acetic acid and H₃PO₄ was achieved within 30 and 60 min, respectively. This revealed that H₃PO₄ has negative impact on the reaction rate and quantity of the proline-ninhydrin complex formed. In brief, our proline quantification protocol involves reaction of a 1-ml proline sample with 2 ml of 1.25 % ninhydrin in glacial acetic acid at 100 °C for 30 min, followed by recording absorbance of the proline-ninhydrin condensation product in the reaction mixture itself at 508 nm. Amongst proline quantification protocols known till date, our protocol is the most simple, rapid, reliable, cost-effective, and eco-friendlier.     

Lignified and nonlignified fiber cables in the lacunae of <Emphasis Type="Italic">Typha angustifolia</Emphasis>

The leaves of Typha are noteworthy in terms of their mechanical properties. We determined the mechanical properties of the fiber cables within the leaf. We found that in vegetative plants, the lignified fiber cables isolated from the leaf sheath and nonlignified fiber cables isolated from the leaf blade of Typha angustifolia differ in their diameter, swelling capacity, Young’s modulus, tensile strength, and break load. These differing properties are related to their contributions to stability in the two regions of the leaf.     

Salt-stress-responsive chloroplast proteins in <Emphasis Type="Italic">Brassica juncea</Emphasis> genotypes with contrasting salt tolerance and their quantitative PCR analysis

Brassica juncea is mainly cultivated in the arid and semi-arid regions of India where its production is significantly affected by soil salinity. Adequate knowledge of the mechanisms underlying the salt tolerance at sub-cellular levels must aid in developing the salt-tolerant plants. A proper functioning of chloroplasts under salinity conditions is highly desirable to maintain crop productivity. The adaptive molecular mechanisms offered by plants at the chloroplast level to cope with salinity stress must be a prime target in developing the salt-tolerant plants. In the present study, we have analyzed differential expression of chloroplast proteins in two Brassica juncea genotypes, Pusa Agrani (salt-sensitive) and CS-54 (salt-tolerant), under the effect of sodium chloride. The chloroplast proteins were isolated and resolved using 2DE, which facilitated identification and quantification of 12 proteins that differed in expression in the salt-tolerant and salt-sensitive genotypes. The identified proteins were related to a variety of chloroplast-associated molecular processes, including oxygen-evolving process, PS I and PS II functioning, Calvin cycle and redox homeostasis. Expression analysis of genes encoding differentially expressed proteins through real time PCR supported our findings with proteomic analysis. The study indicates that modulating the expression of chloroplast proteins associated with stabilization of photosystems and oxidative defence plays imperative roles in adaptation to salt stress.     

Localization of the human neonatal Fc receptor (FcRn) in human nasal epithelium

The airway epithelium is a central player in the defense against pathogens including efficient mucociliary clearance and secretion of immunoglobulins, mainly polymeric IgA, but also IgG. Pulmonary administration of therapeutic antibodies on one hand, and intranasal immunization on the other, are powerful tools to treat airway infections. In either case, the airway epithelium is the primary site of antibody transfer. In various epithelia, bi-polar transcytosis of IgG and IgG immune complexes is mediated by the human neonatal Fc receptor, FcRn, but FcRn expression in the nasal epithelium had not been demonstrated, so far. We prepared affinity-purified antibodies against FcRn α-chain and confirmed their specificity by Western blotting and immunofluorescence microscopy. These antibodies were used to study the localization of FcRn α-chain in fixed nasal tissue. We here demonstrate for the first time that ciliated epithelial cells, basal cells, gland cells, and endothelial cells in the underlying connective tissue express the receptor. A predominant basolateral steady state distribution of the receptor was observed in ciliated epithelial as well as in gland cells. Co-localization of FcRn α-chain with IgG or with early sorting endosomes (EEA1-positive) but not with late endosomes/lysosomes (LAMP-2-positive) in ciliated cells was observed. This is indicative for the presence of the receptor in the recycling/transcytotic pathway but not in compartments involved in lysosomal degradation supporting the role of FcRn in IgG transcytosis in the nasal epithelium.     

Plant cytochrome P450s: nomenclature and involvement in natural product biosynthesis

Cytochrome P450s constitute the largest family of enzymatic proteins in plants acting on various endogenous and xenobiotic molecules. They are monooxygenases that insert one oxygen atom into inert hydrophobic molecules to make them more reactive and hydro-soluble. Besides for physiological functions, the extremely versatile cytochrome P450 biocatalysts are highly demanded in the fields of biotechnology, medicine, and phytoremediation. The nature of reactions catalyzed by P450s is irreversible, which makes these enzymes attractions in the evolution of plant metabolic pathways. P450s are prime targets in metabolic engineering approaches for improving plant defense against insects and pathogens and for production of secondary metabolites such as the anti-neoplastic drugs taxol or indole alkaloids. The emerging examples of P450 involvement in natural product synthesis in traditional medicinal plant species are becoming increasingly interesting, as they provide new alternatives to modern medicines. In view of the divergent roles of P450s, we review their classification and nomenclature, functions and evolution, role in biosynthesis of secondary metabolites, and use as tools in pharmacology.     

Deciphering the roles of acyl-CoA-binding proteins in plant cells

Lipid trafficking is vital for metabolite exchange and signal communications between organelles and endomembranes. Acyl-CoA-binding proteins (ACBPs) are involved in the intracellular transport, protection, and pool formation of acyl-CoA esters, which are important intermediates and regulators in lipid metabolism and cellular signaling. In this review, we highlight recent advances in our understanding of plant ACBP families from a cellular and developmental perspective. Plant ACBPs have been extensively studied in Arabidopsis thaliana (a dicot) and to a lesser extent in Oryza sativa (a monocot). Thus far, they have been detected in the plasma membrane, vesicles, endoplasmic reticulum, Golgi apparatus, apoplast, cytosol, nuclear periphery, and peroxisomes. In combination with biochemical and molecular genetic tools, the widespread subcellular distribution of respective ACBP members has been explicitly linked to their functions in lipid metabolism during development and in response to stresses. At the cellular level, strong expression of specific ACBP homologs in specialized cells, such as embryos, stem epidermis, guard cells, male gametophytes, and phloem sap, is of relevance to their corresponding distinct roles in organ development and stress responses. Other interesting patterns in their subcellular localization and spatial expression that prompt new directions in future investigations are discussed.     

Effects of fusaric acid treatment on the protocorm-like bodies of <Emphasis Type="Italic">Dendrobium</Emphasis> sonia-28

Dendrobium sonia-28 is a popular orchid hybrid due to its flowering recurrence and dense inflorescences. Unfortunately, it is being decimated by fungal diseases, especially those caused by Fusarium proliferatum. In this study, selection of F. proliferatum-tolerant protocorm-like bodies (PLBs) was carried out by assessing the effects of differing concentrations of fusaric acid (FA). PLBs were cultured on Murashige and Skoog (MS) medium supplemented with 0.05 to 0.2 millimolar (mM) concentrations of FA. Higher concentrations of FA increased mortality of PLBs and reduced their growth. The survival rate for 0.05 mM FA was 20 % but only 1 % at the highest dose of 0.2 mM. Additionally, two different size ranges of PLBs were investigated, and growth increased more at lower FA concentrations for larger PLBs, whilst the growth rate of smaller PLBs was inhibited at an FA concentration of 0.2 mM. Histological examination using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses disclosed severe cell wall and organelle damage, as well as stomatal closure in PLBs treated with the high FA concentrations. Reductions in plantlet growth were much greater at the highest concentrations of FA. Some randomly amplified polymorphic DNA (RAPD) markers clearly discriminated between selected and non-selected variants of Dendrobium sonia-28, showing different banding patterns for each FA concentration and specific bands for selected and control plants.     

Bone Growth Dynamics of the Facial Skeleton and Mandible in <Emphasis Type="Italic">Gorilla gorilla</Emphasis> and <Emphasis Type="Italic">Pan troglodytes</Emphasis>

Adult craniofacial morphology results from complex processes that involve growth by bone modelling and interactions of skeletal components to keep a functional and structural balance. Previous analyses of growth dynamics in humans revealed critical changes during late ontogeny explaining particular morphological features in our species. Data on bone modelling patterns from other primate species could help us to determine whether postnatal changes in the growth dynamics of the craniofacial complex are human specific or are shared with other primates. However, characterizations of bone modelling patterns through ontogeny in non-human hominids are scarce and restricted to isolated data on facial and mandibular regions. In the present study, we analyse the bone modelling patterns in an ontogenetic series of Pan and Gorilla to infer the growth dynamics of their craniofacial complex during postnatal development. Our results show that both Pan troglodytes and Gorilla gorilla are characterized by species-specific bone modelling patterns indicative of a mainly forward growth direction during postnatal development. Both species show minor but consistent ontogenetic changes in the distribution of bone modelling fields in specific regions of the face and mandible, in contrast to other regions which show more constant bone modelling patterns. In addition, we carry out a preliminary integrative study merging histological and geometric morphometric data. Both approaches yield highly complementary data, each analysis providing details on specific growth dynamics unavailable to the other. Moreover, geometric morphometric data show that ontogenetic variation in the modelling pattern of the mandibular ramus may be linked to sexual dimorphism.     

Partial Opsin Sequences Suggest UV-Sensitive Vision is Widespread in Caudata

Ultraviolet (UV) vision exists in several animal groups. Intuitively, one would expect this trait to be favoured in species living in bright environments, where UV light is the most present. However, UV sensitivity, as deduced from sequences of UV photoreceptors and/or ocular media transmittance, is also present in nocturnal species, raising questions about the selective pressure maintaining this perceptual ability. Amphibians are among the most nocturnal vertebrates but their visual ecology remains poorly understood relative to other groups. Perhaps because many of these species breed in environments that filter out a large part of UV radiation, physiological and behavioural studies of UV sensitivity in this group are scarce. We investigated the extent of UV vision in Caudata, the order of amphibians with the most nocturnal habits. We could recover sequences of the UV sensitive SWS1 opsin in 40 out of 58 species, belonging to 6 families. In all of these species, the evidence suggests the presence of functional SWS1 opsins under purifying selection, potentially allowing UV vision. Interestingly, most species whose opsin genes failed to amplify exhibited particular ecological features that could drive the loss of UV vision. This likely wide distribution of functional UV photoreceptors in Caudata sheds a new light on the visual ecology of amphibians and questions the function of UV vision in nocturnal animal species.