Response of lentil to VA Mycorrhizal inoculation and plant available P levels of unsterile soils

Summary Responses of lentil in unsterile soils at low, medium and high levels of plant available soil P toGlomus fasciculatum inoculation were evaluated. It was observed that growth, dry matter accumulation, nodulation, and nitrogen fixation were considerably improved in VAM inoculated plants over uninoculated control at low and medium levels of plant available soil P.     

In vitro regeneration of plants from hypocotyl segments of Amaranthus paniculatus

Hypocotyl segments, 5 to 8 mm length from 4 to 7 day old seedlings, callused on B₅ medium supplemented with Kn (0.5 ppm) and NAA (0.1 ppm). Even without transfer, shoots were formed in such cultures. About 20% of the cultures produced multiple shoots. In medium with 1 ppm each of Kn and NAA direct shoots were formed at one end of the hypocotyl segment and callusing was initiated at the other end. The plants obtained in either medium formed roots and could be transferred to soil for further growth.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxy acetic acid - GA₃ gibberellic acid - Kn Kinetin - NAA naphthalene acetic acid     

Relationship between calcium and agar on vitrification and shoot-tip necrosis of quince (Cydonia oblonga Mill.) shoots in vitro

Shoot-tip cultures of Quince C (Cydonia oblonga Mill.) initiated on Murashige & Skoog (MS) medium containing 5 M BA and 0.6% Phytagar showed both shoot-tip necrosis and severe vitrification. Culturing explants on medium containing 1.2% Phytagar and Ca levels of 3 mM (MS medium), 18 mM and 30 mM showed a decrease in growth with increasing medium Ca levels, being especially severe at 30 mM. The Ca content of the explants increased linearly with increasing medium Ca. Culturing explants on medium containing 3 mM, 9 mM, and 18 mM Ca at 0.6, 0.9, and 1.2% agar resulted in reduction in growth, shoot-tip necrosis, and vitrification when either factor was increased. The reduction in shoot-tip necrosis could be accounted for primarily by an increase in medium Ca levels but may also be affected by a change in explant growth. Increasing Ca concentration in the medium resulted in a linear increase in explant K, Ca, Mg, and B levels and a decrease in Mn and Na. Although increasing medium Ca or agar levels reduced vitrification, it is unclear whether they were the direct cause of the reduction in vitrification or whether this response was an effect of the reduction in culture fresh weight.Approved by publication by the Director, West Virginia Agriculture anf Forestry Experimental Station as Scientific Article No. 2199     

Structural insights into thermostable direct hemolysin of Vibrio parahaemolyticus using single-particle cryo-EM

Thermostable direct hemolysin (TDH) is a ~19 kDa, hemolytic pore-forming toxin from the gram-negative marine bacterium Vibrio parahaemolyticus, one of the causative agents of seafood-borne acute gastroenteritis and septicemia. Previous studies have established that TDH exists as a tetrameric assembly in physiological state; however, there is limited knowledge regarding the molecular arrangement of its disordered N-terminal region (NTR)—the absence of which has been shown to compromise TDH's hemolytic and cytotoxic abilities. In our current study, we have employed single-particle cryo-electron microscopy to resolve the solution-state structures of wild-type TDH and a TDH construct with deletion of the NTR (NTD), in order to investigate structural aspects of NTR on the overall tetrameric architecture. We observed that both TDH and NTD electron density maps, resolved at global resolutions of 4.5 and 4.2 Å, respectively, showed good correlation in their respective oligomeric architecture. Additionally, we were able to locate extra densities near the pore opening of TDH which might correspond to the disordered NTR. Surprisingly, under cryogenic conditions, we were also able to observe novel supramolecular assemblies of TDH tetramers, which we were able to resolve to 4.3 Å. We further investigated the tetrameric and inter-tetrameric interaction interfaces to elaborate upon the key residues involved in both TDH tetramers and TDH super assemblies. Our current structural study will aid in understanding the mechanistic aspects of this pore-forming toxin and the role of its disordered NTR in membrane interaction.     

Organogenesis in callus derived from stem and leaf tissues of apple and cherry rootstocks

Callus formation from stem internodes of the apple rootstocks M.9, M.25, M.26, M.27 and the cherry rootstock Colt, and from pith of Nicotiana tabacum cv. Wisconsin 38 was initiated on 4 -naphthaleneacetic acid (NAA)-based media (2.0–10.0 mg1^-1). Transfer of callus to corresponding media lacking NAA allowed regeneration of shoots from callus of M.25, M.27, Colt and tobacco but not of M.9 and M.26. With M.25 phloroglucinol (PG) depressed regeneration from 30 to 10% and no regeneration was observed in cultures grown in the presence of casein hydrolysate (CH) and glutathione (GSH).Organogenesis was also obtained from leaf discs of M.27 employing 6-benzyl-aminopurine (BAP) at 5.0mg 1^-1 and 2,4-dichlorophenoxy acetic acid (2,4-D) at 0.1 mg1^-1. The regenerated shoots have been multiplied and rooted.Organogenesis also occurred in M.26 from small (1–2mm), green, compact embryoid-like structures derived from stem and leaf surfaces of excised axillary shoots. These structures differentiated into shoots at a low frequency (< 1%) on media containing BAP (1.0mg1^-1) and indole-3-butyric acid (IBA) (0.1 mg1^-1) and could also be micropropagated by subsequent axillary shoot proliferation.     

Linkage relationships between regulatory and structural gene loci involved in zein synthesis in maize

Summary The synthesis of at least 15 zein polypeptides is under the control of regulatory gene loci. One of these, Opaque-2 (chromosome 7, position 16) strongly reduces the zein accumulation without modifying the zein molecular components. The linkage relationship between the regulatory gene 02 and the 5 structural loci (Zp1, Zp2, Zp3, Zp6, Zp12) segregating with sample Mendelian ratios have been studied. Zp1, Zp2, Zp3 are closely linked to each other; moreover this gene cluster is located on chromosome 7 at 5.5 cM from the Opaque-2 locus. The structural loci Zp6 and Zp12 are not linked with each other, with the 02 locus or with Zp1, Zp2, Zp3. From our data it follows that the zein structural genes are located in at least three positions on the maize genome. The scattering in the genome of the genes controlled by the Opaque-2 locus suggests a transacting role for this regulatory element.     

Changes in carbohydrates and lipids during embryonic development ofAntheraea mylitta (Lepidoptera)

Changes in carbohydrate and lipid metabolism during embryonic development inAntheraea mylitta were studied. While carbohydrates were metabolized during early embryogenesis, lipids were catabolised at the later stages. A significant increase in both total carbohydrates and glycogen on days 5 and 6 suggested the concurrent occurrence of both gluconeogenesis and glycogenesis. As the development of the embryo proceeds, both lipids and carbohydrates were utilised, resulting in the increase in the concentration of citrate, pyruvate and lactate.     

Cryopreservation of Cymbidium eburneum Lindl. and C. hookerianum Rchb. f., two threatened and vulnerable orchids via encapsulation–dehydration

A successful cryopreservation protocol for the long-term conservation of protocorms of two threatened and vulnerable orchids, Cymbidium eburneum Lindl. and Cymbidium hookerianum Rchb. f., was developed using encapsulation–dehydration. Protocorms were osmoprotected in liquid Murashige and Skoog medium (MS) containing 0.7 M sucrose for 20 h at 25?±?2°C on a rotary shaker, and incorporated into an encapsulation matrix [consisting of 3% (w/v) sodium alginate and 100 mM CaCl₂]. The encapsulated protocorms, which were desiccated in a laminar airflow cabinet for 6 h, were able to withstand cryostorage in liquid nitrogen. Maximum regeneration into complete plantlets (72% for C. eburneum and 70% for C. hookerianum) of the cryostored, encapsulated protocorms was obtained using MS medium containing 3% sucrose and 0.8% agar. Using this protocol of cryopreservation, long-term preservation for ex situ conservation of these two threatened orchids can be accomplished.     

Ratiometric Ca+2 measurement in human recombinant muscarinic receptor subtypes using the Flexstation scanning fluorometer

In modern drug discovery, numerous assay formats are available to screen and quantitate receptor-ligand interactions. Radioactive assays are “gold standard” because they are fast, easy, and reproducible; however, they are hazardous, produce radioactive waste, require special lab conditions, and are expensive on a large scale. Thus, it provides a lot of importance to the “mix & measure” assays that have an optical readout. Fluorescence techniques are likely to be among the most important detection approaches used for high throughput screening due to their high sensitivity and amenability to automation. The aim of the present study was to determine the functional antagonistic affinities of standard muscarinic antagonists in CHO cells over expressing m1, m3, and m5 receptors and to compare them with the respective binding affinities. This study was further extended to elucidate that Ca⁺² measurement assays can serve as a functional screening tool for GPCRs. For this purpose, standard muscarinic receptor antagonists, namely, tolterodine, oxybutynin, and atropine were used. We determined and compard the IC₅₀ values of these three standard inhibitors in fura 2 AM loaded m1, m3, and m5 overexpressing CHO cells and in radioligand binding assay. Both the assays exhibited comparable rank order potencies of the standard inhibitors. This study suggests that Ca⁺² mobilization assays can be an alternate to radioligand binding assays.     

The PPARα agonist fenofibrate suppresses B-cell lymphoma in mice by modulating lipid metabolism

Obesity is associated with an increased risk for malignant lymphoma development. We used Bcr/Abl transformed B cells to determine the impact of aggressive lymphoma formation on systemic lipid mobilization and turnover. In wild-type mice, tumor size significantly correlated with depletion of white adipose tissues (WAT), resulting in increased serum free fatty acid (FFA) concentrations which promote B-cell proliferation in vitro. Moreover, B-cell tumor development induced hepatic lipid accumulation due to enhanced hepatic fatty acid (FA) uptake and impaired FA oxidation. Serum triglyceride, FFA, phospholipid and cholesterol levels were significantly elevated. Consistently, serum VLDL/LDL-cholesterol and apolipoprotein B levels were drastically increased. These findings suggest that B-cell tumors trigger systemic lipid mobilization from WAT to the liver and increase VLDL/LDL release from the liver to promote tumor growth. Further support for this concept stems from experiments where we used the peroxisome proliferator-activated receptor α (PPARα) agonist and lipid-lowering drug fenofibrate that significantly suppressed tumor growth independent of angiogenesis and inflammation. In addition to WAT depletion, fenofibrate further stimulated FFA uptake by the liver and restored hepatic FA oxidation capacity, thereby accelerating the clearance of lipids released from WAT. Furthermore, fenofibrate blocked hepatic lipid release induced by the tumors. In contrast, lipid utilization in the tumor tissue itself was not increased by fenofibrate which correlates with extremely low expression levels of PPARα in B-cells. Our data show that fenofibrate associated effects on hepatic lipid metabolism and deprivation of serum lipids are capable to suppress B-cell lymphoma growth which may direct novel treatment strategies. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.