Sonication-assisted efficient Agrobacterium-mediated genetic transformation of the multipurpose woody desert shrub Leptadenia pyrotechnica

The desert shrub Leptadenia pyrotechnica (Forssk.) Decne (Asclepiadaceae) is an important multipurpose woody species of tropical and sub-tropical arid regions. The shrub’s excellent pharmacological properties, importance in desert afforestation and role in sand dune fixation has been elaborately studied in recent years which make it a potential candidate for genetic manipulation in the global warming scenario. We have developed an Agrobacterium-mediated transformation protocol for L. pyrotechnica using hypocotyl explants from 5 days old seedlings. The reliability of the protocol has been tested by transforming the species with gus and gfp reporter genes separately. Hypocotyl explants were sonicated for 40 s, infected with Agrobacterium suspension of OD₆₀₀ 0.5, co-cultivated in the dark for 2 days at 26 °C in presence of 200 μM acetosyringone. Transgenic plants were obtained after 20–22 weeks at a frequency of 14 and 11 % for gus and gfp reporter genes respectively. Transgenic plants were confirmed by PCR and Southern blots. Expression of the two reporter genes has been tested in different stages of transgenic plant development. No phenotypic differences between the wild-type and transgenic plants were noted. This method will be very helpful to introduce alien genes-of-interest for various biotechnological applications.     

A Novel Monoclonal Antibody disrupting Cell Type Specific Substratum Adhesion of Frog (Xenopus laevis) Epithelial Cells and Endothelial Cells

We isolated a mouse monoclonal antibody (FAD-II) that disrupts cell-substratum adhesion of amphibian ( Xenopus laevis ) epithelial cells and endothelial cells. The effect of the antibody was cell-type specific, and the antibody had no effect on fibroblastic cells while fibronectin peptide blocked cell-substratum adhesion of all the cell types examined. In developing frog embryos, the epitopes recognized by the antibody were detected in pronephrotic ducts and in other tissue cells of embryos (from stage 33/34 afterwards). In adult tissues, the antibody mainly recognized antigens in extracelluar matrices. The antigens recognized by the antibody seems to be novel glycoepitopes in frog cells.     

A 38 base pair insertion in the pro alpha 2(I) collagen gene of a patient with Marfan syndrome

Abnormalities in type I collagen have been recognized in a number of connective tissue disorders. In the Marfan syndrome, an autosomal dominant condition producing a generalized abnormality in connective tissue, no consistent abnormality has been identified, although one individual has been found to have an elongated pro alpha 2(I) collagen chain [Byers et al, Proc Natl Acad Sci USA 78:7745, 1981]. To determine the nature of the alteration in the gene that produced this abnormality, we studied the pro alpha 2(I) gene from this individual by genomic blotting and gene cloning. Genomic mapping studies detected no abnormalities. However, analysis of the cloned segment of the pro alpha 2(I) collagen gene from the Marfan individual indicates that the gene contains a 38 base pair insertion in an intron near the collagenase cleavage site. Although the relationship of this insertion to the protein abnormality is unclear, it may be a useful marker for the diagnosis of the Marfan syndrome.     

Protic solvent effects on the photophysical properties of O=Ti(IV)TSPP: photoinduced electron transfer.

The photophysical properties of oxotitanium(IV)meso-tetra(4-sulfonatophenyl) porphyrin (O=Ti(IV)TSPP) have been investigated in water and methanol by laser spectroscopic techniques. The fluorescence emission spectrum of O=Ti(IV)TSPP in methanol exhibits two strong emission bands at 610 and 670 nm at room temperature with the decay time of ca. 310 +/- 10 ps and the rise time shorter than 30 ps, in contrast to the extremely weak emission with the decay time of ca. 27 +/- 4 ps in water, indicating that the fluorescence emissive states are different in the two solvents as supported by the solvent dependences of the excitation spectrum. The transient Raman spectra of O=Ti(IV)TSPP in water has been observed to exhibit a remarkable enhancement of phenyl-related mode at 1599 cm(-1), while in methanol, the Raman frequencies of the porphyrin skeletal modes (upsilon2 and upsilon4) are down-shifted without any apparent enhancement of the phenyl-related mode, indicating different interactions of the two solvents with the excited O=Ti(IV)TSPP. These Raman studies reveal that methanol molecule interacts with the photoexcited O=Ti(IV)TSPP more strongly than water, forming the exciplex, O=Ti(IV)TSPP(MeOH)*, suggesting that the two different emissive states are the singlet Franck-Condon state and the exciplex state in methanol and water, respectively. A broad triplet transient absorption of O=Ti(IV)TSPP has been also observed at 480 nm in water as well as in methanol, which is decreased upon addition of methyl viologen (MV2+) with appearance of a new absorption band at 620 nm. This indicates that the photoinduced electron transfer (PET) takes place from the porphyrin to MV2+ in both solvents. The kinetic analysis of the transient absorption band exhibits the PET rate constants of 4.76 x 10(5) s(-1) and 3,03 x 10(4) s(-1) in methanol and water, respectively. All these results infer that the PET takes place from the (d,pi) CT state and the triplet state of the excited porphyrin in methanol and water, respectively.     

Evaluation of intestinal microbiota,short-chain fatty acids,and immunoglobulin a in diversion colitis

It is reported that an increase in aerobic bacteria, a lack of short-chain fatty acids (SCFAs), and immune disorders in the diverted colon are major causes of diversion colitis. However, the precise pathogenesis of this condition remains unclear. The aim of the present study was to examine the microbiota, intestinal SCFAs, and immunoglobulin A (IgA) in the diverted colon. Eight patients underwent operative procedures for colostomies. We assessed the diverted colon using endoscopy and obtained intestinal samples from the diverted colon and oral colon in these patients. We analyzed the microbiota and SCFAs of the intestinal samples. The bacterial communities were investigated using a 16S rRNA gene sequencing method. The microbiota demonstrated a change in the proportion of some species, especially Lactobacillus, which significantly decreased in the diverted colon at the genus level. We also showed that intestinal SCFA values were significantly decreased in the diverted colon. Furthermore, intestinal IgA levels were significantly increased in the diverted colon. This study was the first to show that intestinal SCFAs were significantly decreased and intestinal IgA was significantly increased in the diverted colon. Our data suggest that SCFAs affect the microbiota and may play an immunological role in diversion colitis.     

Hemistepsin A inhibits T0901317-induced lipogenesis in the liver

Hemistepsin A (HsA) is a guaianolide sesquiterpene lactone that inhibits hepatitis and liver fibrosis. We evaluated the effects of HsA on liver X receptor (LXR)-mediated hepatic lipogenesis in vitro and in vivo. Up to 10 μM, HsA did not affect the viability of HepG2 and Huh7 cells. Pretreatment with 5-10 μM HsA significantly decreased the luciferase activity of the LXR response element, which was transactivated by T0901317, GW 3965, and LXRα/retinoid X receptor α overexpression. In addition, it significantly inhibited the mRNA expression of LXRα in HepG2 and Huh7 cells. It also suppressed the expression of sterol regulatory element-binding protein-1c and lipogenic genes and reduced the triglyceride accumulation triggered by T0901317. Intraperitoneal injection of HsA (5 and 10 mg/kg) in mice significantly alleviated the T0901317-mediated increases in hepatocyte diameter and the percentage of regions in hepatic parenchyma occupied by lipid droplets. Furthermore, HsA significantly attenuated hepatic triglyceride accumulation by restoring the impaired expression of LXRα-dependent lipogenic genes caused by T0901317. Therefore, based on its inhibition of the LXRα-dependent signaling pathway, HsA has prophylactic potential for steatosis.     

Intragenic Recombination in the Adh Locus of the Wild Plant Arabidopsis Thaliana

Nucleotide variation in the Adh region of the wild plant Arobidopsis thaliana was analyzed in 17 ecotypes sampled worldwide to investigate DNA polymorphism in natural plant populations. The investigated 2.4-kb Adh region was divided into four blocks by intragenic recombinations between two parental sequence types that diverged 6.3 million years (Myr) ago, if the nucleotide mutation rate μ = 10(-9) is assumed. Within each block, dimorphism of segregating variations was observed with intermediate frequencies, which caused a substantial amount of nucleotide variation in A. thaliana at the species level. The first recombination introduced the divergent variation that resulted in dimorphism in this plant species ~3.3 Myr ago, and three subsequent intragenic recombinations have occurred sporadically in ~1.1-Myr intervals. It was shown that there was only a limited number (six) of sequence types in this species and that no clear association was observed between sequence type and geographic origin. Taken together, these results suggest that A. thaliana has spread over the world only recently. It can be concluded that recombination played an important role in the evolutionary history of A. thaliana, especially through the generation of DNA polymorphism in the natural populations of this plant species.     

Prerequisites in distance running performance of female runners

We investigated which attribute or what combination of attributes would best account for distance running performance of female runners. The subjects were 30 well-trained female distance runners, aged 19 to 23 years. Anthropometric and body composition characteristics, pulmonary function characteristics, blood properties, and cardiorespiratory function characteristics were measured at rest or during submaximal and maximal exercise. Analyses of the data showed that the relationship of oxygen uptake corresponding to lactate threshold (VO2T, ml.kg-1.min-1) with each distance running performance was substantially higher as compared with the relationship of other independent variables including maximal oxygen uptake (VO2max). Furthermore, multiple regression analysis indicated that running performances in 3,000m, 5,000m, and 10,000m are best accounted for by a combination of VO2/LT (X1), fat-free weight (X2), and/or mean corpuscular volume (X3). A multiple regression equation for predicting the 5,000m (Y, s) running performance was formulated as Y = -14.75X1-3. 03X2-5.79X3 + 2282.1. We suggest that VO2max would not stand alone as a decisive factor of distance running success in female runners, and that the distance running performance could be better accounted for by a combination of several attributes relating to lactate threshold, body composition, and/or hematological status. The linear regression of the predicted running performance on the actually measured running performance can be accepted in the range of 986-1197s.