In vitro interactions of histones and α-crystallin

The aggregation of crystallins in lenses is associated with cataract formation. We previously reported that mutant crystallins are associated with an increased abundance of histones in knock-in and knockout mouse models. However, very little is known about the specific interactions between lens crystallins and histones. Here, we performed in vitro analyses to determine whether α-crystallin interacts with histones directly. Isothermal titration calorimetry revealed a strong histone–α-crystallin binding with a K_d of 4 × 10^?7 M, and the thermodynamic parameters suggested that the interaction was both entropy and enthalpy driven. Size-exclusion chromatography further showed that histone–α-crystallin complexes are water soluble but become water insoluble as the concentration of histones is increased. Right-angle light scattering measurements of the water-soluble fractions of histone–α-crystallin mixtures showed a decrease in the oligomeric molecular weight of α-crystallin, indicating that histones alter the oligomerization of α-crystallin. Taken together, these findings reveal for the first time that histones interact with and affect the solubility and aggregation of α-crystallin, indicating that the interaction between α-crystallin and histones in the lens is functionally important.     

In vivo interactions of TGF-β and extracellular matrix

TGF-β, a multifunctional cytokine, plays an important role in embryogenesis and in regulating repair and remodeling following tissue injury. Many of the biological actions of TGF-β are mediated by widespread effects on deposition of extracellular matrix. TGF-β stimulates the synthesis of individual matrix components including proteoglycans, collagens and glycoproteins. TGF-β also blocks matrix degradation by decreasing the synthesis of proteases and increasing the synthesis of protease inhibitors. Finally, TGF-β increases the synthesis of matrix receptors and alters their relative proportions on the surface of cells in a manner that could facilitate adhesion to matrix. All of these events have largely been demonstrated in vitro in cultured cells. In an experimental model of glomerulonephritis we have shown that TGF-β is responsible for the accumulation of pathological matrix in the glomeruli following immunological injury. Furthermore, all three of TGF-β's actions on extracellular matrix—increased synthesis, decreased degradation and modulation of receptors—have now been documented to be involved in matrix deposition in vivo in this model. Administration of the proteoglycan decorin suppressed TGF-β-induced matrix deposition in the nephritic glomeruli, thus confirming a physiological role for decorin as a regulator of TGF-β. Inhibitors of TGF-β may be important future drugs in treating fibrotic diseases caused by overproduction of TGF-β.     

Functional Analyses of Escherichia coli MutS-β Clamp Interaction In Vitro and In Vivo

Escherichia coli MutS is a highly conserved mismatch repair (MMR) protein that plays a key role in recognizing DNA mismatches and the early steps of MMR. Previous studies revealed an interaction between MutS and the replicative protein β clamp, but it remains unclear whether the interaction functions during the process of MMR. In order to provide insight into the significance of this interaction, Far Western, Surface plasmon resonance and cell survival/mutagenesis assays were used to determine its possible influences on the in vitro and in vivo properties of MutS. The results show that a quintuple mutation of MutS residues 812–816 (MutS^βC), or single alanine substitution mutation of MutS residues M813 or L815 completely blocks binding of MutS to β clamp. Wild type β clamp interferes with DNA binding by MutS. When treated with the base analog 2-aminopurine, MutS^βC confers more mutations and less cellular growth rate in the mutS-deficient strain than the wild type MutS. These data indicate that the MutS-β interaction has functional consequences during MMR in E. coli.     

In Vitro Exchange of β-Sitosterol between Erythrocytes and Plasma of Rats

The erythrocytes and plasma of rats were not labeled equally with sterols even after feeding plant sterols for 2 months.

When erythrocytes and plasma were labeled in vivo with radioactive sterols, the in vitro exchange of cholesterol between cells and plasma was considerably greater than that of β-sitosterol. The dependence of the transfer on plasma lecithin : cholesterol acyltransferase was much less with β-sitosterol.

More labeled β-sitosterol existed in high density lipoprotein and less in very-low density and low density lipoproteins than cholesterol, when plasma was labeled in vivo. A similar distribution pattern was observed when plasma was incubated with labeled erythrocytes. These results suggest that an extra ethyl group in the side chain of the molecule substantially influences the metabolic behavior of the sterols.     

In Vitro Culture of Wheat and Genetic Transformation — Retrospect and Prospect

This review focuses on the improvement of wheat (Triticum aestivum L.) via tissue and cell culture and its use in gene transfer techniques. Success of the latter critically depends on the ability to regenerate plants from cells or tissues cultured in vitro. Hence, we have devoted attention to the attempts made so far in obtaining regenerants from diverse explants. Although it is known that immature embryos are the best source for initiating morphogenic cultures, basic information related to the process of differentiation can also be gained by studying less responding tissues. The opportunity provided by anther and microspore culture in wheat improvement and the progress made is also presented.

To enhance tissue culture responses, identification of chromosomes, gene loci, and genes is of cardinal importance. We have also surveyed the progress made in this regard by conventional but incisive plant-breeding techniques. Gene rearrangements in tissue culture leading to the appearance of somaclonal or gametoclonal variation are of interest in selection of useful cell lines. The last part of the review is devoted to work done on transient gene expression and transformation with emphasis on recent developments.     

The Expression and Release of Hsp60 in 6-OHDA Induced In Vivo and In Vitro Models of Parkinson’s Disease

In Parkinson’s disease, dopaminergic neuron damage/death causes the release of soluble substances that are selectively toxic to neighboring/additional dopaminergic neurons through the activation of microglia. Hsp60 can be released from injured cells of central nervous system to activate microglia. However, its expression and role in Parkinson’s disease has not been well understood. Here, we performed a 6-OHDA treated Parkinson’s disease model in adult rats. Western blot analysis showed a time-course expression of Hsp60, which decreased gradually and then rose back. Immunofluorescence staining showed that Hsp60 was decreased in dopaminergic neuron, and most Hsp60 located on the surface of activated microglia. Furthermore, in cellular Parkinson’s disease model, Hsp60 was obviously detected in the culture supernatants after 6-OHDA treatment, and a concomitant decrease in cell extracts. Taken together, our results suggested that Hsp60 could be released extracellularly to activate microglia in Parkinson’s disease model.     

Eudragit-Based Nanosuspension of Poorly Water-Soluble Drug: Formulation and In Vitro–In Vivo Evaluation

The present study was performed to investigate potential of Eudragit RLPO-based nanosuspension of glimepiride (Biopharmaceutical Classification System class II drug), for the improvement of its solubility and overall therapeutic efficacy, suitable for peroral administration. Nanoprecipitation method being simple and less sophisticated was optimized for the preparation of nanosuspension. Physicochemical characteristics of nanosuspension in terms of size, zeta potential, polydispersity index, entrapment efficiency (% EE) and in vitro drug release were found within their acceptable ranges. The size of the nanoparticles was most strongly affected by agitation time while % EE was more influenced by the drug/polymer ratio. Differential scanning calorimetry and X-ray diffraction studies provided evidence that enhancement in solubility of drug resulted due to change in crystallinity of drug within the formulation. Stability study revealed that nanosuspension was more stable at refrigerated condition with no significant changes in particle size distribution, % EE, and release characteristics for 3 months. In vivo studies were performed on nicotinamide–streptozotocin-induced diabetic rat models for pharmacokinetic and antihyperglycaemic activity. Nanosuspension increased maximum plasma concentration, area under the curve, and mean residence time values significantly as compared to aqueous suspension. Oral glucose tolerance test and antihyperglycaemic studies demonstrated plasma glucose levels were efficiently controlled in case of nanosuspension than glimepiride suspension. Briefly, sustained and prolonged activity of nanosuspensions could reduce dose frequency, decrease drug side effects, and improve patient compliance. Therefore, glimepiride nanosuspensions can be expected to gain considerable attention in the treatment of type 2 diabetes mellitus due to its improved therapeutic activity.KEY WORDS: diabetes mellitus, glimepiride, nanoprecipitation, poloxamer, sustained release     

Fabrication and In Vitro/In Vivo Performance of Mucoadhesive Electrospun Nanofiber Mats Containing α-Mangostin

This study aimed to fabricate mucoadhesive electrospun nanofiber mats containing α-mangostin for the maintenance of oral hygiene and reduction of the bacterial growth that causes dental caries. Synthesized thiolated chitosan (CS-SH) blended with polyvinyl alcohol (PVA) was selected as the mucoadhesive polymer. α-Mangostin was incorporated into the CS-SH/PVA solution and electrospun to obtain nanofiber mats. Scanning electron microscopy, differential scanning calorimetry, X-ray diffraction, and tensile strength testing were used to characterize the mats. The swelling degree and mucoadhesion were also determined. The nanofiber mats were further evaluated regarding their α-mangostin content, in vitro α-mangostin release, antibacterial activity, cytotoxicity, in vivo performance, and stability. The results indicated that the mats were in the nanometer range. The α-mangostin was well incorporated into the mats, with an amorphous form. The mats showed suitable tensile strength, swelling, and mucoadhesive properties. The loading capacity increased when the initial amount of α-mangostin was increased. Rapid release of α-mangostin from the mats was achieved. Additionally, a fast bacterial killing rate occurred at the lowest concentration of nanofiber mats when α-mangostin was added to the mats. The mats were less cytotoxic after use for 72 h. Moreover, in vivo testing indicated that the mats could reduce the number of oral bacteria, with a good mouth feel. The mats maintained the amount of α-mangostin for 6 months. The results suggest that α-mangostin-loaded mucoadhesive electrospun nanofiber mats may be a promising material for oral care and the prevention of dental caries.KEY WORDS: dental caries, mucoadhesive property, nanofibers, thiolated chitosan, α-mangostin     

Mechanism of Adsorption and Eclipse of Bacteriophage φX174 III. Comparison of the Activation Parameters for the In Vitro and In Vivo Eclipse Reactions With Mutant and Wild-Type Virus

In a starvation buffer containing 10(-3) M divalent cations, phiX174 undergoes viral eclipse above 20 C when attached to intact host cells. An in vitro structural transition that is similar to that observed in this in vivo eclipse reaction occurs over the same temperature range in 0.1 M CaCl(2) (pH 7.2). Since both reactions result in a loss of infectivity, their kinetics have been compared in this report. Both exhibit a biphasic first-order loss in PFU that is a result of two competing first-order processes. However, a single type of heterogeneity in the population of virions is not the basis for both competing slower reactions. The Arrhenius plots of the faster components show that the in vitro eclipse reaction has the same activation energy of 35 kcal/mol (ca. 1.47 x 10(5) J/mol) as the in vivo reaction but a 10-fold lower Arrhenius preexponential factor. This is further evidence that certain features of the in vivo mechanism are retained in the in vitro reaction. In the case of the slower components, the in vitro reaction has an activation energy of 37 kcal/mol (1.55 x 10(5) J/mol), whereas that of the in vivo reaction is only 5 kcal/mol (2.1 x 10(4) J/mol). A similar analysis has been performed on a cold-sensitive eclipse mutant of phiX174. In vivo, the mutation is expressed by a two- to three-fold lower Arrhenius preexponential factor for both components of the eclipse reaction when compared to wt virus. The activation energies for both components are the same as wt virus. These results suggest that the mechanism of the eclipse reaction can be operationally divided into two aspects, each subject to mutational alteration.     

In vitro seed culture and seedling development of Calopogon tuberosus

A major obstacle to native orchid production is difficulty in seed germination. Culture media and light effects on seed germination of Calopogon tuberosus var. tuberosus, a native orchid with horticultural potential, were studied. Culture media included Knudson C, Malmgren modified terrestrial orchid, and PhytoTechnology orchid seed sowing. Effects of 8 weeks continual darkness, 8 weeks 16-h photoperiod, 2 weeks dark followed by 6 weeks 16-h photoperiod, 4 weeks dark followed by 4 weeks 16-h photoperiod, and 6 weeks dark followed by 2 weeks 16-h photoperiod were examined. Percent seed germination was highest on Knudson C after 8 weeks culture; however, seedling development was enhanced on PhytoTechnology seed sowing medium during 8 weeks culture under a 16-h photoperiod. This suggests that while KC and darkness promoted seed germination, P723 and light enhanced further seedling development. Seedlings of C. tuberosus readily acclimated to greenhouse conditions.     

In situ Protection Management and Conservation Study of Some Medicinal Plants

In situ study on eleven commercially important species viz;Adiantum capillus veneris L,Bergenia ciliata (Haw) Sternb,Colchicum luteum Baker,Polygonum amplexicaule D.Den,Cuminum cyminum L,Dioscorea deltoidea Wall Kunth,L Morchella esculenta L,Paeonia emodi Wall ex H Kf,Podophyllum hexandrum (Royle) Chatt & Mukh,Valeriana wallichii DC and Viola serpens Wall ex Roxb was conducted in four locations viz; Malam (1 400 to 2 000?m),Bargin (1 700 to 2 300?m),Biakand (1 500 to 2 100?m) and Shinko (2 100 to 2 700?m).The data was recorded from both protected and unprotected sites of each site.Each site had 3 altitudinal sampling point.The density, herbage coverage and fresh biomass were determined in each locations for every species.All the investigated parameters generally showed an increase of 3 to 6 times over unprotected sites in each locations.Morchella esculenta,Dioscorea deltoidea,Colchicum luteum and Podophyllum hexandrum were absent in all unprotected sites while other species had low values in these stes.The investigated parameters of Colchicum luteum,Bergenia ciliata,Paeonioa emodi,Dioscorea deltoidea and Podophyllum hexandrum generally increased with the increasing elevation.Soil analysis,soil and air temperatures were recorded for each site.The air and soil temperature were slightly higher in open areas than in the protected site and showed decrease with increasing elevation.While the soil fertility was relatively high in protected sites as compared to unprotected area.The study shows that protection promotes the growth, distribution and occurrence of medicinal plants.It is possible with the participation of local communities to conserve these resources.     

Convergence of Amyloid-β and Tau Pathologies on Mitochondria In Vivo

The histopathological characteristics of Alzheimer’s disease (AD) are amyloid-β (Aβ) containing plaques and neurofibrillary tangles (NFTs) as well as neuronal and synaptic loss. Until today, the underlying mechanisms of the interplay of plaques and tangles remained unresolved. There is increasing evidence that mitochondrial dysfunction might be a possible link, as revealed by studies in several APP and tau transgenic mouse models. Recently, we examined mitochondrial function in a novel triple transgenic mouse model (pR5/APP/PS2)—^tripleAD mice—that combines both pathologic features of the disease in brain. Using comparative, quantitative proteomics (iTRAQ) and mass spectroscopy, we found a massive deregulation of 24 proteins, of which one third were mitochondrial proteins mainly related to complexes I and IV of the oxidative phosphorylation system (OXPHOS). Remarkably, deregulation of complex I was related to tau, whereas deregulation of complex IV was Aβ dependent, both at the protein and activity levels. The ^tripleAD mice showed synergistic effects of Aβ and tau already at the age of 8 months, resulting in a depolarized mitochondrial membrane potential. At 12 months, the strongest defects on OXPHOS, synthesis of ATP and reactive oxygen species, were exhibited in the ^tripleAD mice, again emphasizing synergistic, age-associated effects of Aβ and tau in impairing mitochondria. This review highlights the convergence of Aβ and tau on mitochondria and establishes a molecular link in AD pathology in vivo.     

In situ activation of β-galactosidase of Kluyveromyces bulgaricus resting cells by sodium and potassium phosphates and chlorides

Summary Incubation of Kluyveromyces bulgaricus in sodium and potassium salts led to in vivo activation of -galactosidase. The activation reaction was relatively slow since, at 37°C, it took 30 min to come to completion. The reaction was irreversible and was favoured by high salt concentrations with chlorides proving to be more efficient than phosphates. After incubation in KCl, the final activity obtained was 1.49 U/mg dry yeast and this represented a 10-fold increase in activity compared with the control value measured in ammonium phosphate. Hydrolysis of onitrophenyl--galactoside (ONPG) was insensitive to inhibitors of the transport systems and energy metabolism. There results suggest that K. bulgaricus resting cells take up substrates and ions that readily influence -galactosidase activity.     

Subcellular localization of proflavine derivative and induction of oxidative stress—In vitro studies

Acridines have been studied for several decades because of their numerous biological effects, especially anticancer activity. Recently, cytotoxicity of novel acridine derivatives, 3,6-bis((1-alkyl-5-oxo-imidazolidin-2-yliden)imino)acridine hydrochlorides (AcrDIMs), was confirmed for leukemic cell lines [Bioorg. Med. Chem. 2011, 19, 1790]. The mechanism of action of the most cytotoxic hexyl-AcrDIM was studied in this paper focusing attention on a subcellular distribution of the drug. Accumulation of hexyl-AcrDIM in mitochondria was confirmed after labeling mitochondria with MitoRED using ImageStream Imaging Flow Cytometer. The derivative significantly decreased intracellular ATP level (reduction of ATP level was decreased by vitamin E), and induced oxidative stress (ROS production detected by DHE assay) as well as cell cycle arrest in the S-phase (flow cytometry analysis) already after short-time incubation and induction of apoptosis. Cytotoxicity of hexyl-AcrDIM is closely connected with induction of oxidative stress in cells.     

Root-To-Shoot Signalling: Assessing The Roles of ‘Up’ In the Up and Down World of Long-Distance Signalling In Planta

An important mediator of shoot physiological processes can be the supply of signal molecules (other than water and nutrients) from the root system. Root-to-shoot signalling is often considered to be important in regulating shoot growth and water use when soil conditions change without any demonstrable change in shoot water or nutrient status. Changes in xylem sap composition are often thought to be synonymous with changes in root-to-shoot signalling, even though there is considerable re-cycling of compounds between xylem and phloem. Techniques used to collect xylem sap are reviewed. Elucidating the roles of putative root signal molecules in planta has usually taken priority over identifying the sources of signal molecules in xylem sap. The roles of several signal molecules are considered. This choice is selective, and the failure of known signals to account for observed physiological changes in some systems has lead to the conclusions that other novel signals can be important. The efficacy of a given signal molecule can depend on the shoot water and nutrient status, as demonstrated by variation in stomatal responses to abscisic acid. If such variation is widespread in crop species, this may have implications for the increasing intentional use of root-to-shoot signals to modify crop water use and shoot architecture. Research into root-to-shoot signalling may become increasingly reductionist, in trying to evaluate the contribution of root signals versus local processes to observed physiological changes. However, future challenges are to successfully integrate this basic research into improved crop production systems.     

In vitro and in vivo molecular imaging of estrogen receptor α and β homo- and heterodimerization: exploration of new modes of receptor regulation

Estrogen receptor (ER) biology reflects the actions of estrogens through the two receptors, ERα and ERβ, although little is known regarding the preference for formation of ER homo- vs. heterodimers, and how this is affected by the level of ligand occupancy and preferential ligand affinity for one of the ER subtypes. In this report, we use a split optical reporter-protein complementation system to demonstrate the physical interaction between ERα and ERβ in response to different ER ligands in cells and, for the first time, by in vivo imaging in living animals. The genetically encoded reporter vectors constructed with the ligand-binding domains of ERα and ERβ, fused to split firefly or Renilla luciferase (Fluc or hRluc) fragments, were used for this study. This molecular proteomic technique was used to detect ERα/ERα or ERβ/ERβ homodimerization, or ERα/ERβ heterodimerization induced by ER subtype-selective and nonselective ligands, and selective ER modulators (SERM), as well as in dimers in which one mutant monomer was unable to bind estradiol. The SERM-bound ERα and ERβ form the strongest dimers, and subtype-preferential homodimerization was seen with ERα-selective ligands (methyl piperidino pyrazole/propyl pyrazole triol) and the ERβ-selective ligands (diarylpropionitrile/tetrahydrochrysene/genistein). We also demonstrated that a single ligand-bound monomer can form homo- or heterodimers with an apo-monomer. Xenografts of human embryonic kidney 293T cells imaged in living mice by bioluminescence showed real-time ligand induction of ERα/ERβ heterodimerization and reversal of dimerization upon ligand withdrawal. The results from this study demonstrate the value of the split luciferase-based complementation system for studying ER-subtype interactions in cells and for evaluating them in living animals by noninvasive imaging. They also probe what combinations of ERα and ERβ dimers might be the mediators of the effects of different types of ER ligands given at different doses.