Nature of interference potential of leaf debris of Ageratum conyzoides

The present study investigated the allelopathic interference of leaf debris of Ageratum conyzoides (billy goat weed; Asteraceae)—a weed of cultivated land—against rice (Oryza sativa). Seedling length and dry weight of rice were significantly reduced (16–20%) in soil from A. conyzoides infested fields compared to the soil from an area devoid of the weed. It indicated the presence of certain phytotoxins in the A. conyzoides infested soil. To explore the possible contribution of the weed in releasing these phytotoxins, growth studies involving leaf debris extracts and amended soils (prepared by incorporating leaf debris—5, 10, 20 g kg⁻¹ soil, w/w, or its extracts—0.5%, 1.0% and 2.0%, v/v) were conducted. The growth of rice was severely inhibited in A. conyzoides leaf debris- and debris extract-amended soils compared to unamended control soil. A significant amount of water-soluble phenolics, the potent phytotoxins, was found in the A. conyzoides infested soil, leaf debris, and debris-amended soils. These phenolics were identified as gallic acid, coumalic acid, protocatechuic acid, catechin and p-hydroxybenzoic acid. Among these, protocatechuic acid was in the maximum amount (35.72%) followed by coumalic acid (33.49%) and these two accounted for >69% of total phenolic compounds. Further, there was a significant increase in the available nutrient content in soil amended with A. conyzoides leaf debris thus ruling out the possibility of any resource depletion upon residue incorporation and their negative role in causing growth reduction. Based on the observations, the present study concludes that leaf debris of A. conyzoides deleteriously affects the early growth of rice by releasing water-soluble phenolic acids into the soil environment and not through soil nutrient depletion.     

Calreticulin transacylase: Genesis,mechanism of action and biological applications

Our earlier investigations have identified a unique enzyme in the endoplasmic reticulum (ER) termed Acetoxy Drug: Protein Transacetylase (TAase) catalyzing the transfer of acetyl group from polyphenolic acetates (PA) to certain receptor proteins (RP). An elegant assay procedure for TAase was developed based on the inhibition of glutathione S-transferase (GST) due to acetylation by a model acetoxycoumarin, 7, 8-Diacetoxy-4-methylcoumarin (DAMC). TAase purified from various mammalian tissue microsomes to homogeneity exhibited a molecular weight (M.wt) of 55 kDa. Further, by N-terminal sequencing TAase was identified as Calreticulin (CR), a multifunctional Ca²⁺-binding protein in ER lumen. The identity of TAase with CR was evidenced by proteomics studies such as immunoreactivity with anti-CR antibody and mass spectrometry. This function of CR was termed Calreticulin transacetylase (CRTAase). CRTAase was also found to mediate the transfer of acetyl group from DAMC to RP such as NADPH Cytochrome c Reductase (CYPR) and Nitric Oxide Synthase (NOS). The autoacetylation of purified human placental CRTAase concomitant with the acetylation of RP by DAMC was observed. CRTAase activity was found to be inhibited by Ca²⁺. Our investigations on the individual domains (N, P and C) of CR from a nematode Haemonchus contortus revealed that the P-domain alone was found to possess CRTAase activity. Based on the observation that the autoacetylated CR was a stable intermediate in the CRTAase catalyzed protein acetylation by PA, a putative mechanism was proposed. Further, CRTAase was also found capable of transferring propionyl group from a propoxy derivative of polyphenol, 7,8-Dipropoxy-4-methylcoumarin (DPMC) to RP and concomitant autopropionylation of CR was encountered. Hence, CRTAase was assigned the general term Calreticulin Transacylase. Also, CRTAase was found to act upon the biological acyl group donors, acetyl CoA and propionyl CoA. CRTAase mediated modulation of specific functional proteins by way of acylation was exploited to elicit the biological applications of PA.     

The Quest to Understand the Basis and Mechanisms that Control Expression of Introduced Transgenes in Crop Plants

We discuss mechanisms and factors that influence levels and stability of expressed heterologous proteins in crop plants. We have seen substantial progress in this field over the past two decades in model experimental organisms such as Arabidopsis and tobacco. There is no question such studies have resulted in furthering our understanding of key processes in the plant cell and the elaboration of sophisticated models to explain underlying mechanisms that might influence the fate, levels and stability of expression of recombinant heterologous proteins in plants. However, very often, such information is not applicable outside these laboratory experimental models. In order to generate a knowledge basis that can be used to achieve high levels and stability of heterologous proteins in relevant crop plants it is imperative to perform such studies on the target crops. With this in mind, we discuss key elements of the process at the DNA, RNA and protein levels. We believe it is essential to discuss recombinant protein production in crops in a holistic manner in order to develop a comprehensive knowledge base that will in turn serve plant biotechnology applications well.Key Words: transgene expression, protein trafficking, silencing mechanisms, chromatin remodeling, crop plants     

Associations between Retinal Markers of Microvascular Disease and Cognitive Impairment in Newly Diagnosed Type 2 Diabetes Mellitus: A Case Control Study

Objective

To investigate associations between retinal microvascular changes and cognitive impairment in newly diagnosed type 2 diabetes mellitus.

Design

Case control study.

Setting

A primary care cohort with newly diagnosed type 2 diabetes mellitus.

Methods

For this analysis, we compared 69 cases with lowest decile scores (for the cohort) on the Modified Telephone Interview for Cognitive Status and 68 controls randomly selected from the remainder of the cohort. Retinal images were rated and the following measures compared between cases and controls: retinal vessel calibre, arterio-venous ratio, retinal fractal dimension, and simple and curvature retinal vessel tortuosity.

Results

Total and venular (but not arteriolar) simple retinal vessel tortuosity levels were significantly higher in cases than controls (t = 2.45, p = 0.015; t = 2.53, p = 0.013 respectively). The associations persisted after adjustment for demographic factors, retinopathy, neuropathy, obesity and blood pressure. There were no other significant differences between cases and controls in retinal measures.

Conclusions

A novel association was found between higher venular tortuosity and cognitive impairment in newly diagnosed type 2 diabetes mellitus. This might be accounted for by factors such as hypoxia, thrombus formation, increased vasoendothelial growth factor release and inflammation affecting both the visible retinal and the unobserved cerebral microvasculature.     

A quantitative assay to measure chromosome stability in Schizosaccharomyces pombe

Summary The fidelity of mitotic chromosome transmission in Schizosaccharomyces pombe was estimated quantitatively by using cycloheximide resistance as a means to select cells that had undergone chromosome loss or nondisjunction. We aimed to investigate the connection between recombination and mitotic chromosome stability. A number of mutants defective in mitotic recombination such as cdc17-L16, rec59-72, and rec50-25 were tested and in these an approximately ten fold elevation of mitotic haploidization rate was found compared with controls. Our data suggest that recombination is important in controlling the maintenance of chromosomes during mitosis.     

M26 Recombinational Hotspot and Physical Conversion Tract Analysis in the Ade6 Gene of Schizosaccharomyces Pombe

At the ade6 locus of Schizosaccharomyces pombe flanking markers have been introduced as well as five silent restriction site polymorphisms: four in the 5' upstream region and one in the middle of the gene. The mutations ade6-706, ade6-M26 (both at the 5' end) and ade6-51 (middle of the gene) were used as partners for crosses with the 3' mutation ade6-469. From these three types of crosses, wild-type recombinants were selected and analyzed genetically to assess association with crossing-over and physically to determine conversion tract lengths. The introduced restriction site polymorphisms (five vs. only one) neither influenced the pattern of recombinant types nor the distribution of conversion tracts. The hotspot mutation M26 enhances crossing-over and conversion to the same proportion. M26 not only stimulates conversion at the 5' end, but does this also (to a lower extent) at the 3' end of ade6 at a distance of more than 1 kb. The majority of meiotic conversion tracts are continuous and postmeiotic segregation of polymorphic sites is rare. Conversion tracts are slightly shorter with M26 in comparison with its control 706. The mean minimal length of tracts varies from 670 bp (M26) to 890 bp (706) to 1290 bp (51). It is concluded that M26 acts as an initiation site of recombination or enhances initiation of recombination. M26 does not act by termination of conversion. A region of recombination initiation exists at the 5' end of the ade6 gene also in the absence of the ade6-M26 hotspot mutation.     

Real-time Monitoring of Ligand-receptor Interactions with Fluorescence Resonance Energy Transfer

FRET is a process whereby energy is non-radiatively transferred from an excited donor molecule to a ground-state acceptor molecule through long-range dipole-dipole interactions¹. In the present sensing assay, we utilize an interesting property of PDA: blue-shift in the UV-Vis electronic absorption spectrum of PDA (Figure 1) after an analyte interacts with receptors attached to PDA^2,3,4,7. This shift in the PDA absorption spectrum provides changes in the spectral overlap (J) between PDA (acceptor) and rhodamine (donor) that leads to changes in the FRET efficiency. Thus, the interactions between analyte (ligand) and receptors are detected through FRET between donor fluorophores and PDA. In particular, we show the sensing of a model protein molecule streptavidin. We also demonstrate the covalent-binding of bovine serum albumin (BSA) to the liposome surface with FRET mechanism. These interactions between the bilayer liposomes and protein molecules can be sensed in real-time. The proposed method is a general method for sensing small chemical and large biochemical molecules. Since fluorescence is intrinsically more sensitive than colorimetry, the detection limit of the assay can be in sub-nanomolar range or lower⁸. Further, PDA can act as a universal acceptor in FRET, which means that multiple sensors can be developed with PDA (acceptor) functionalized with donors and different receptors attached on the surface of PDA liposomes.