Analysis of phenotypic and microsatellite-based diversity of maize landraces in India,especially from the North East Himalayan region

The maize landraces in the North East Himalayan (NEH) region in India, especially in the Sikkim state, are morphologically highly diverse. The present study provides details of phenotypic and molecular characterization of a set of 48 selected maize landrace accessions, including the ‘Sikkim Primitives’ which have a unique habit of prolificacy (5–9 ears on a single stalk). Multi-location phenotypic evaluation of these 48 accessions revealed significant genetic variability for grain yield and its components, leading to identification of several promising accessions. Cluster analysis and PCA using nine morpho-agronomic characters clearly separated ‘Sikkim Primitives’ from the rest of the accessions. PCA revealed two principal components describing 90% of the total variation, with hundred kernel weight, ear length, ear diameter, number of kernels per ear and flowering behaviour forming the most discriminatory traits. The accessions were genotyped using 42 microsatellite or simple sequence repeat (SSR) markers using a ‘population bulk DNA fingerprinting strategy’, with allele resolution using an automated DNA Sequencer. The study revealed a high mean number of alleles per SSR locus (13.0) and high Polymorphism Information Content (PIC) value of 0.60. The analysis also led to identification of 163 private/unique alleles, differentiating 44 out of 48 accessions. Six highly frequent SSR alleles were detected at different loci (phi014, phi062, phi090, umc1266, umc1367 and umc2250) with individual frequencies ≥0.75. Some of these SSR loci were reported to tag specific genes/QTL for some important traits, indicating that chromosomal regions harboring these SSR alleles were not selectively neutral. Cluster analysis using Rogers’ genetic distance also revealed distinct genetic identity of the ‘Sikkim Primitives’ from the rest of the accessions in India, including Sikkim. Mantel’s test revealed significant and positive correlation between the phenotypic and molecular genetic dissimilarity matrices. The study was the first to portray the patterns of phenotypic and molecular diversity in the maize landraces from the NEH region in India.     

Assessing photosynthetic efficiency in an experimental mangrove canopy using remote sensing and chlorophyll fluorescence

This study examined the ability of the photochemical reflectance index (PRI) to track changes in effective quantum yield (Δ F/F _m ′), non-photochemical quenching (NPQ), and the xanthophyll cycle de-epoxidation (DPS) in an experimental mangrove canopy. PRI was correlated with (Δ F/F _m ′) and NPQ over the 4-week measurement period and over the diurnal cycle. The normalised difference vegetation index (NDVI) was not correlated with any aspect of photochemical efficiency measured using chlorophyll fluorescence or xanthophyll pigments. This study demonstrated that photochemical adjustments were responsible for controlling the flow of energy through the photosynthetic apparatus in this mangrove forest canopy rather than canopy structural or chlorophyll adjustments.     

Developing an Extended Genomic Engineering Approach Based on Recombineering to Knock-in Heterologous Genes to <Emphasis Type="Italic">Escherichia coli</Emphasis> Genome

Most existing genomic engineering protocols for manipulation of Escherichia coli are primarily focused on chromosomal gene knockout. In this study, a simple but systematic chromosomal gene knock-in method was proposed based on a previously developed protocol using bacteriophage λ (λ Red) and flippase–flippase recognition targets (FLP–FRT) recombinations. For demonstration purposes, DNA operons containing heterologous genes (i.e., pac encoding E. coli penicillin acylase and palB2 encoding Pseudozyma antarctica lipase B mutant) engineered with regulatory elements, such as strong/inducible promoters (i.e., P _trc and P _araB ), operators, and ribosomal binding sites, were integrated into the E. coli genome at designated locations (i.e., lacZYA, dbpA, and lacI-mhpR loci) either as a gene replacement or gene insertion using various antibiotic selection markers (i.e., kanamycin and chloramphenicol) under various genetic backgrounds (i.e., HB101 and DH5α). The expression of the inserted foreign genes was subjected to regulation using appropriate inducers [isopropyl β-d-1-thiogalactopyranoside (IPTG) and arabinose] at tunable concentrations. The developed approach not only enables more extensive genomic engineering of E. coli, but also paves an effective way to “tailor” plasmid-free E. coli strains with desired genotypes suitable for various biotechnological applications, such as biomanufacturing and metabolic engineering.     

Cell Wall Regeneration in Bangia atropurpurea (Rhodophyta) Protoplasts Observed Using a Mannan-Specific Carbohydrate-Binding Module

The cell wall of the red alga Bangia atropurpurea is composed of three unique polysaccharides (β-1,4-mannan, β-1,3-xylan, and porphyran), similar to that in Porphyra. In this study, we visualized β-mannan in the regenerating cell walls of B. atropurpurea protoplasts by using a fusion protein of a carbohydrate-binding module (CBM) and green fluorescent protein (GFP). A mannan-binding family 27 CBM (CBM27) of β-1,4-mannanase (Man5C) from Vibrio sp. strain MA-138 was fused to GFP, and the resultant fusion protein (GFP–CBM27) was expressed in Escherichia coli. Native affinity gel electrophoresis revealed that GFP–CBM27 maintained its binding ability to soluble β-mannans, while normal GFP could not bind to β-mannans. Protoplasts were isolated from the fronds of B. atropurpurea by using three kinds of bacterial enzymes. The GFP–CBM27 was mixed with protoplasts from different growth stages, and the process of cell wall regeneration was observed by fluorescence microscopy. Some protoplasts began to excrete β-mannan at certain areas of their cell surface after 12 h of culture. As the protoplast culture progressed, β-mannans were spread on their entire cell surfaces. The percentages of protoplasts bound to GFP–CBM27 were 3%, 12%, 17%, 29%, and 25% after 12, 24, 36, 48, and 60 h of culture, respectively. Although GFP–CBM27 bound to cells at the initial growth stages, its binding to the mature fronds was not confirmed definitely. This is the first report on the visualization of β-mannan in regenerating algal cell walls by using a fluorescence-labeled CBM.     

Operation of packed-bed immobilized cell reactor featuring active β-galactosidase inclusion body-containing recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis> cells

In this study, we developed a packed-bed immobilized cell reactor containing active β-gal (β-galactosidase) inclusion body (IB)-containing Escherichia coli (E. coli) cells in alginate beads. This packed-bed reactor was operated using a substrate feed solution 0.72 ∼ 38.4 mM ONPG (o-nitrophenyl-β-D-galactoside) prepared in Z buffer supplemented with chloroform and 0.1% SDS (sodium dodecyl sulfate). The production rate of ONP (o-nitrophenol) in the reactor containing cells that were incubated with α-MG (α-methyl D-glucospyranoside) or D-fucose after induction was superior to those prepared with cells that were not incubated with α-MG or D-fucose. The ONP production rate was increased proportionally with ONPG concentration in the substrate feed up to a concentration of 38.4 mM. However, as the ONPG concentration was increased in the substrate feed solution, galactose inhibition inside the alginate beads was increased. This most likely occurred due to problems with diffusion. In addition, partial breakage of alginate beads was observed during the later periods of operation. In this study, we demonstrated that active β-gal IB-containing E. coli cells were sustained in the immobilized cell reactor during operation. Particularly, these findings demonstrate the feasibility of using active IBs in an enzymatic reaction without the need for any purification step. In addition, we showed that these IB-containing cells could be directly used in an immobilized reactor.     

Plasmid-Related Resistance to Cefoxitin in Species of the Bacteroides fragilis Group Isolated from Intestinal Tracts of Calves

Species of the Bacteroides fragilis group are considered the most common anaerobe in human and animal infections and also harbor plasmids conferring resistance to several antibiotics. In this study, resistance to cefoxitin, plasmid profile and β-lactamase production in species of the B. fragilis group isolated from intestinal tracts of calves were evaluated. One hundred sixty-one B. fragilis group bacteria isolated from calves with and without diarrhea were analyzed. Cefoxitin susceptibility was performed using an agar dilution method, β-lactamase production by using a nitrocefin method, and plasmid extraction by using a commercial kit. Minimal inhibitory concentration values for cefoxitin ranged from 32 to > 512 μg/ml, and 47 bacteria (29.2%) were resistant to cefoxitin (breakpoint 16 μl). Only seven isolates harbored plasmids varying from 6.0 to 5.0 kb, and a 5.5-kb plasmid in B. vulgatus Bd26e and B. fragilis Bc5j might be related to cefoxitin resistance. β-lactamase was detected in 33 (70.2%) isolates. The cepA gene was observed in total DNA and in the 5.5-kb plasmid. The plasmid presence in organisms isolated from cattle may be important in ecologic terms, and it needs further study.     

Improvement of protein production in lactic acid bacteria using 5′-untranslated leader sequence of slpA from Lactobacillus acidophilus

The 5′-untranslated leader sequence (UTLS) of the slpA gene from Lactobacillus acidophilus contributes to mRNA stabilization by producing a 5′ stem and loop structure, and a high-level expression system for the lactic acid bacteria was developed using the UTLS in this study. A plasmid, which expresses α-amylase under the control of the ldh promoter, was constructed by integrating the core promoter sequence with the UTLS. The role of the UTLS in increasing the copies of the α-amylase mRNA was proved by measuring α-amylase activity in the culture supernatant and the relative expression of α-amylase mRNA was determined by the quantitative real-time PCR analysis. Moreover, several expression systems were constructed by combining the core promoter sequence with the UTLS or with the partially deleted UTLS and the expression level was evaluated. The use of the UTLS led to the success in improving α-amylase expression in the two strains of Lactobacillus casei and Lactococcus lactis. The current study showed that the improvement in protein production using the UTLS could be applied to the expression system in the lactic acid bacteria.     

A novel association of a polymorphism in the first intron of adiponectin gene with type 2 diabetes, obesity and hypoadiponectinemia in Asian Indians

Adiponectin is an adipose tissue specific protein that is decreased in subjects with obesity and type 2 diabetes. The objective of the present study was to examine whether variants in the regulatory regions of the adiponectin gene contribute to type 2 diabetes in Asian Indians. The study comprised of 2,000 normal glucose tolerant (NGT) and 2,000 type 2 diabetic, unrelated subjects randomly selected from the Chennai Urban Rural Epidemiology Study (CURES), in southern India. Fasting serum adiponectin levels were measured by radioimmunoassay. We identified two proximal promoter SNPs (−11377C→G and −11282T→C), one intronic SNP (+10211T→G) and one exonic SNP (+45T→G) by SSCP and direct sequencing in a pilot study (n = 500). The +10211T→G SNP alone was genotyped using PCR-RFLP in 4,000 study subjects. Logistic regression analysis revealed that subjects with TG genotype of +10211T→G had significantly higher risk for diabetes compared to TT genotype [Odds ratio 1.28; 95% Confidence Interval (CI) 1.07–1.54; P = 0.008]. However, no association with diabetes was observed with GG genotype (P = 0.22). Stratification of the study subjects based on BMI showed that the odds ratio for obesity for the TG genotype was 1.53 (95%CI 1.3–1.8; P < 10⁻⁷) and that for GG genotype, 2.10 (95% CI 1.3–3.3; P = 0.002). Among NGT subjects, the mean serum adiponectin levels were significantly lower among the GG (P = 0.007) and TG (P = 0.001) genotypes compared to TT genotype. Among Asian Indians there is an association of +10211T→G polymorphism in the first intron of the adiponectin gene with type 2 diabetes, obesity and hypoadiponectinemia.     

Response surface methodology in media optimization for production of β-carotene from <Emphasis Type="Italic">Daucus carota</Emphasis>

Plants are a valuable source of a vast array of chemical compounds including fragrances, flavours, food additives, colours, natural sweeteners, industrial feedstocks, anti-microbials and pharmaceuticals. The present study reports on application of Response Surface Methodology (RSM) in media optimization for suspension culture for the production of β-carotene. Growth kinetics of carrot cells in suspension culture has been carried out to understand the relationship between growth and β-carotene formation. The maximum production of β-carotene obtained using the optimized medium was 13.614 μg/g dry weight cell mass. The μ (specific growth rate) and t _d (doubling time) were found to be higher for 20 g DW/l inoculum size.     

Invariants of the anthropometrical proportions

The work introduces a generalized interpretation of a modulor as a scale of segment proportions of human anthropometrical modules (extremities and body). Aims of study: (1) to reason the new idea of a “growth modulor”; (2) using the modern empirical data, to prove the validity of a principle of linear similarity for anthropometrical segments; (3) to specify a system of invariants for constitutional anthropometrics.     

不同松树种间杂交类型的可育性分析

探究不同松树种间杂交可育性,为松树种间杂交育种亲本选配提供依据,进而为松树的杂种优势研究积累材料.该研究利用随机区组设计,通过92个松树种间杂交组合与20个半同胞对照,分析9种松树种间杂交类型子代的平均球果产种数、平均球果产种量、百粒重、发芽率与成苗率5项育性指标,综合球果期、种期与苗期3个阶段的表现,利用模糊数学隶属...     

A fast algal bioassay for assessment of copper toxicity in water using <Emphasis Type="Italic">Euglena gracilis</Emphasis>

A rapid, sensitive algal bioassay was investigated to monitor copper toxicity using photosynthetic activity and motility parameters as end points in the photosynthetic flagellate Euglena gracilis. Effects on motility parameters were determined using the automatic bioassay ECOTOX. Photosynthetic efficiency was measured using chlorophyll fluorescence by means of a fast, noninvasive pulse amplitude modulated fluorometer. These parameters were assessed regarding their effectiveness as end points for short- (0–24 h) and long-term (1–5 days) toxicity tests. The model organism showed significant responses to the tested concentrations at both motility and photosynthesis examined levels. EC₅₀ values for movement parameters immediately after the cells were mixed with copper are 19.09 (300.4 μM), 20.4 (312 μM) and 23.09 (363.35 μM) mg L⁻¹ for motility, r-value and velocity, respectively. The current study proofs that Euglena is a convenient biotest organism where motility parameters show rapid sensitive responses to copper (direct exposure to copper). Also, the photosynthetic parameters appear to be appropriate for acute and chronic toxicity tests for higher and lower copper concentrations, respectively.     

Mass–density relationship changes along salinity gradient in <Emphasis Type="Italic">Suaeda salsa</Emphasis> L.

Whether and why the biomass–density (M–N) scaling relationship varies along environmental gradients were continuously debated in theoretical ecology. In this study, how soil salinity stress affects on the M–N scaling relationship was investigated by using Suaeda salsa L. in beach of Dongtai, Jiangsu Province, China. The results showed that the exponent of the scaling relationship (b) of low salinity level (−1.259) was smaller than that of middle salinity level (−1.025), which in turn was smaller than that high salinity level (−0.698). The plant height–crown radius (H–r) scaling exponents (∂) decreased with increasing salinity stress, while the canopy coverage–density (C–N) scaling exponents (β) showed an inverse trend. The predict data (b) based on ∂ and β by using the geometric model were statistically indistinguishable from their observed values for the three salinity levels. Moreover, two resources utilization parameters (l mean from root to leaf, a total area of leaves) of metabolic theory, photosynthetic rate, and water-use efficiency were more advantageous to Suaeda salsa L. of high stress than to those of low salinity. Therefore, it was implied that the changes of M–N relationship along salinity gradients may be resulted from their different geometric morphologies and resource utilization in response to salinity stress.     

The effect of body weight on some welfare indicators in feedlot cattle in a hot environment

Heat stress has important effects on the welfare of livestock. The effects of heat stress in cattle include changes in biological functions and behaviors. The aim of this study was to investigate the behavioral differences between light and heavy feedlot cattle reared in a hot environment. Sixteen male Holstein feedlot cattle were allocated to light (353.8 ± 15.5 kg, n = 8) and heavy (737.1 ± 15.8 kg, n = 8) groups according to their live weight and were kept in a semi-open feedlot barn. The individual behavioral response variables measured were standing, lying, feeding, drinking, ruminating, locomotor activity and elimination (urinating and defecating). The effects of group, day, observation time, replicate and all interactions were included in an explanatory statistical (GLM) model. The data were analyzed using the PROC GLM procedure of SAS. Overall, the heavy cattle spent more time standing (P < 0.001), lying (P < 0.001), and eliminating (P < 0.05) compared to the light group. In contrast, the light group spent more time eating, drinking and ruminating (P < 0.001). Locomotor activity did not differ significantly between groups (P > 0.05). During the day, heavy cattle spent more time standing (at 1600 hours) and less time eating in comparison with the light cattle (P < 0.001) (at 1300 and 1600 hours). Light and heavy feedlot cattle behaved differently in a hot environment. The findings of the study indicate that the welfare of the heavy Holstein feedlot cattle was impacted negatively when the ambient temperature was high (at 1300 hours).     

A GIS-based empirical model for vegetation prediction in Lefka Ori, Crete

The aim of the study was to establish and verify a predictive vegetation model for plant community distribution in the alti-Mediterranean zone of the Lefka Ori massif, western Crete. Based on previous work three variables were identified as significant determinants of plant community distribution, namely altitude, slope angle and geomorphic landform. The response of four community types against these variables was tested using classification trees analysis in order to model community type occurrence. V-fold cross-validation plots were used to determine the length of the best fitting tree. The final 9node tree selected, classified correctly 92.5% of the samples. The results were used to provide decision rules for the construction of a spatial model for each community type. The model was implemented within a Geographical Information System (GIS) to predict the distribution of each community type in the study site. The evaluation of the model in the field using an error matrix gave an overall accuracy of 71%. The user’s accuracy was higher for the Crepis–Cirsium (100%) and Telephium–Herniaria community type (66.7%) and relatively lower for the Peucedanum–Alyssum and Dianthus–Lomelosia community types (63.2% and 62.5%, respectively). Misclassification and field validation points to the need for improved geomorphological mapping and suggests the presence of transitional communities between existing community types.     

The β-tubulin gene as a molecular phylogenetic marker for classification and discrimination of the <Emphasis Type="Italic">Saccharomyces</Emphasis> <Emphasis Type="Italic">sensu stricto</Emphasis> complex

The Saccharomyces sensu stricto complex comprises seven very closely related species. In this study, we compared the use of two different phylogenetic markers, the 26S rDNA and β-tubulin genes, for discriminating phylogenetic relationships among Saccharomyces sensu stricto strains using sequencing as well as RFLP methods. The average sequence similarity for the β-tubulin gene (90.0%) among seven strains was significantly less than that for 26S rDNA (98.6%). This result demonstrates that β-tubulin gene sequences provided higher resolution than 26S rDNA sequences. Species-specific restriction profiles of the Saccharomyces strains were obtained by cutting them with the Tsp509I enzyme. Our data indicate that phylogenetic relationships between these strains are best resolved using sequencing or RFLP analysis of the β-tubulin gene. The β-tubulin gene sequence data reported in this paper appear in the GenBank nucleotide sequence database with the following accession numbers: FJ238316–FJ238341.     

The Effect of Selenium Supplementation in the Prevention of DNA Damage in White Blood Cells of Hemodialyzed Patients: A Pilot Study

Patients with chronic kidney disease (CKD) have an increased incidence of cancer. It is well known that long periods of hemodialysis (HD) treatment are linked to DNA damage due to oxidative stress. In this study, we examined the effect of selenium (Se) supplementation to CKD patients on HD on the prevention of oxidative DNA damage in white blood cells. Blood samples were drawn from 42 CKD patients on HD (at the beginning of the study and after 1 and 3 months) and from 30 healthy controls. Twenty-two patients were supplemented with 200 μg Se (as Se-rich yeast) per day and 20 with placebo (baker's yeast) for 3 months. Se concentration in plasma and DNA damage in white blood cells expressed as the tail moment, including single-strand breaks (SSB) and oxidative bases lesion in DNA, using formamidopyrimidine glycosylase (FPG), were measured. Se concentration in patients was significantly lower than in healthy subjects (P < 0.0001) and increased significantly after 3 months of Se supplementation (P < 0.0001). Tail moment (SSB) in patients before the study was three times higher than in healthy subjects (P < 0.01). After 3 months of Se supplementation, it decreased significantly (P < 0.01) and was about 16% lower than in healthy subjects. The oxidative bases lesion in DNA (tail moment, FPG) of HD patients at the beginning of the study was significantly higher (P < 0.01) compared with controls, and 3 months after Se supplementation it was 2.6 times lower than in controls (P < 0.01). No changes in tail moment was observed in the placebo group. In conclusion, our study shows that in CKD patients on HD, DNA damage in white blood cells is higher than in healthy controls, and Se supplementation prevents the damage of DNA.     

Over-expressed estrogen receptor-α up-regulates hTNF-α gene expression and down-regulates β-catenin signaling activity to induce the apoptosis and inhibit proliferation of LoVo colon cancer cells

TAK-778 has been shown to stimulate osteogenesis both in vitro and in vivo. However, the mechanism by which TAK-778 exerts its effects is still unclear. There is evidence that TAK-778 acts via estrogen-receptor (ER)-mediated signaling; this study therefore aimed to investigate the roles that ERα, ERβ, and membrane ER play in the osteogenic effect of TAK-778. To this end, human bone marrow mesenchymal cells were cultured with TAK-778 in the presence of either ICI182,780 (ERα and ERβ antagonist) or MPP (ERα antagonist) or PD98059 (an extracellular-regulated kinase inhibitor that acts on the membrane ER pathway). The following parameters were evaluated: cell proliferation, collagen content, alkaline phosphatase (ALP) activity and bone-like formation. Data were compared using ANOVA. The effect of TAK-778 on expression of ERα and ERβ was investigated by immunolabeling. In order to investigate whether TAK-778 binds to ER, an ER binding assay was performed. Both immunolabeling and binding assays were conducted using cells from human alveolar bone. The osteogenic effect of TAK-778 was inhibited by ICI182,780 and MPP; however, it was not affected by PD98059. The expression of both ERα and ERβ was not affected by TAK-778. The competition curve obtained from the binding assay using TAK-778 showed maximal displacement when 10⁻⁵ M TAK-778 was used. This study's results show that TAK-778 enhances osteoblast differentiation through an ERα-dependent pathway by binding to this receptor and not by increasing the expression of ER. (Mol Cell Biochem xxx: 1–9, 2005)     

Nitrogen Controls on Fine Root Substrate Quality in Temperate Forest Ecosystems

Nitrogen controls on fine root substrate quality (that is, nitrogen and carbon-fraction concentrations) were assessed using nitrogen availability gradients in the Harvard Forest chronic nitrogen addition plots, University of Wisconsin Arboretum, Blackhawk Island, Wisconsin, and New England spruce-fir transect. The 27 study sites encompassed within these four areas collectively represented a wide range of nitrogen availability (both quantity and form), soil types, species composition, aboveground net primary production, and climatic regimes. Changes in fine root substrate quality among sites were most frequently and strongly correlated with nitrate availability. For the combined data set, fine root nitrogen concentration increased (adjusted R ² = 0.46, P < 0.0001) with increasing site nitrate availability. Fine root “extractive” carbon-fraction concentrations decreased (adjusted R ² = 0.32, P < 0.0002), “acid-soluble” compounds increased (adjusted R ² = 0.35, P < 0.0001), and the “acid-insoluble” carbon fraction remained relatively high and stable (combined mean of 48.7 ± 3.1% for all sites) with increasing nitrate availability. Consequently, the ratio of acid-insoluble C–total N decreased (adjusted R ² = 0.40, P < 0.0001) along gradients of increasing nitrate availability. The coefficients of determination for significant linear regressions between site nitrate availability and fine root nitrogen and carbon-fraction concentrations were generally higher for sites within each of the four study areas. Within individual study sites, tissue substrate quality varied between roots in different soil horizons and between roots of different size classes. However, the temporal variation of fine root substrate quality indices within specific horizons was relatively low. The results of this study indicate that fine root substrate quality increases with increasing nitrogen availability and thus supports the substrate quality component of a hypothesized conceptual model of nitrogen controls on fine root dynamics that maintains that fine root production, mortality, substrate quality, and decomposition increase with nitrogen availability in forest ecosystems in a manner that is analogous to foliage.