Synthesis,in vitro and in vivo studies of Gd-DTPA-XDA-D1-Glc(OH) complex as a new potential MRI contrast agent

A new type of dendritic molecules Gd-DTPA-XDA-D1-Glc(OH), which work as a functionalized ligand coordinating gadolinium(III) ion at the center of their frameworks with two glucose moieties on the molecular surfaces, were readily synthesized with high yield. The structures were established by IR, ¹H, ¹³C NMR, and mass spectral studies. Its bio-distribution patterns were evaluated on rats.     

Dimeric and monomeric organization of photosystem II. Distribution of five distinct complexes in the different domains of the thylakoid membrane

The supramolecular organization of photosystem II (PSII) was characterized in distinct domains of the thylakoid membrane, the grana core, the grana margins, the stroma lamellae, and the so-called Y100 fraction. PSII supercomplexes, PSII core dimers, PSII core monomers, PSII core monomers lacking the CP43 subunit, and PSII reaction centers were resolved and quantified by blue native PAGE, SDS-PAGE for the second dimension, and immunoanalysis of the D1 protein. Dimeric PSII (PSII supercomplexes and PSII core dimers) dominate in the core part of the thylakoid granum, whereas the monomeric PSII prevails in the stroma lamellae. Considerable amounts of PSII monomers lacking the CP43 protein and PSII reaction centers (D1-D2-cytochrome b559 complex) were found in the stroma lamellae. Our quantitative picture of the supramolecular composition of PSII, which is totally different between different domains of the thylakoid membrane, is discussed with respect to the function of PSII in each fraction. Steady state electron transfer, flash-induced fluorescence decay, and EPR analysis revealed that nearly all of the dimeric forms represent oxygen-evolving PSII centers. PSII core monomers were heterogeneous, and a large fraction did not evolve oxygen. PSII monomers without the CP43 protein and PSII reaction centers showed no oxygen-evolving activity.     

Survivability of parthenogenetic embryos following in vivo transfer in naturally synchronized Capra hircus

The present study was conducted to see the in vivo developmental potency of caprine parthenogenetic embryos generated in a modified way. The good quality caprine oocytes were matured in presence of cytochalasin B (CCB) and then activated by 7% ethanol followed by 2 mM 6-dimethyl amino purine (6-DMAP) and embryo development was recorded. Early stage parthenogenetic embryos (two to four cells) were surgically transferred in recipients (10). The pregnancy diagnosis was done by nonreturn to oestrus, ultrasonography (USG), and progesterone estimation. The levels of progesterone were above normal values (1 ng/ml) of pregnancy and fall below the level of pregnancy just before retuned to oestrus. Progesterone profile revealed that out of ten recipients (G1–G10), four goats (G1, G2, G3, and G5) returned to oestrus after 43?±?7.29 (Mean?±?SE) d of embryo transfer and six goats (G4, G6, G7, G8, G9, and G10) did not return to cycle even after 70 d of embryo transfer. In three recipients (G4, G5, and G6), the USG on day 40 revealed that there was fluid filled uterine body with solid fetus-like structure. These might be dead fetus and had started resorption. The progesterone profile also corroborated the assumption of pregnancy in these animals. Authors believe that this may be the first report on in vivo diploid parthenogenetic embryo development in caprine species.     

Myostatin induces cachexia by activating the ubiquitin proteolytic system through an NF-kappaB-independent, FoxO1-dependent mechanism

Myostatin, a transforming growth factor-beta (TGF-beta) super-family member, has been well characterized as a negative regulator of muscle growth and development. Myostatin has been implicated in several forms of muscle wasting including the severe cachexia observed as a result of conditions such as AIDS and liver cirrhosis. Here we show that Myostatin induces cachexia by a mechanism independent of NF-kappaB. Myostatin treatment resulted in a reduction in both myotube number and size in vitro, as well as a loss in body mass in vivo. Furthermore, the expression of the myogenic genes myoD and pax3 was reduced, while NF-kappaB (the p65 subunit) localization and expression remained unchanged. In addition, promoter analysis has confirmed Myostatin inhibition of myoD and pax3. An increase in the expression of genes involved in ubiquitin-mediated proteolysis is observed during many forms of muscle wasting. Hence we analyzed the effect of Myostatin treatment on proteolytic gene expression. The ubiquitin associated genes atrogin-1, MuRF-1, and E214k were upregulated following Myostatin treatment. We analyzed how Myostatin may be signaling to induce cachexia. Myostatin signaling reversed the IGF-1/PI3K/AKT hypertrophy pathway by inhibiting AKT phosphorylation thereby increasing the levels of active FoxO1, allowing for increased expression of atrophy-related genes. Therefore, our results suggest that Myostatin induces cachexia through an NF-kappaB-independent mechanism. Furthermore, increased Myostatin levels appear to antagonize hypertrophy signaling through regulation of the AKT-FoxO1 pathway.     

Isolation and identification of an endophytic strain of Fusarium oxysporum producing podophyllotoxin from Juniperus recurva

Podophyllotoxin, a well-known naturally occurring aryltetralin lignan occurs in few plant species that is used as a precursor for the chemical synthesis of the anticancer drugs like etoposide, teniposide and etopophose phosphate. The availiability of this lignan is becoming increasingly limited because of the scarce occurance of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. This paper reports first time the production of podophyllotoxin by an endophytic fungus Fusarium oxysporum isolated from the medicinal plant Juniperus recurva. Further confirmation and quantification of podophyllotoxin was performed by HPLC, LC-MS, and LC-MS/MS.     

Cross-species comparison of Drosophila male accessory gland protein genes

Drosophila melanogaster males transfer seminal fluid proteins along with sperm during mating. Among these proteins, ACPs (Accessory gland proteins) from the male's accessory gland induce behavioral, physiological, and life span reduction in mated females and mediate sperm storage and utilization. A previous evolutionary EST screen in D. simulans identified partial cDNAs for 57 new candidate ACPs. Here we report the annotation and confirmation of the corresponding Acp genes in D. melanogaster. Of 57 new candidate Acp genes previously reported in D. melanogaster, 34 conform to our more stringent criteria for encoding putative male accessory gland extracellular proteins, thus bringing the total number of ACPs identified to 52 (34 plus 18 previously identified). This comprehensive set of Acp genes allows us to dissect the patterns of evolutionary change in a suite of proteins from a single male-specific reproductive tissue. We used sequence-based analysis to examine codon bias, gene duplications, and levels of divergence (via dN/dS values and ortholog detection) of the 52 D. melanogaster ACPs in D. simulans, D. yakuba, and D. pseudoobscura. We show that 58% of the 52 D. melanogaster Acp genes are detectable in D. pseudoobscura. Sequence comparisons of ACPs shared and not shared between D. melanogaster and D. pseudoobscura show that there are separate classes undergoing distinctly dissimilar evolutionary dynamics.     

Molecular packing and intermolecular interactions in two structural polymorphs of N-palmitoylethanolamine, a type 2 cannabinoid receptor agonist

The molecular structure, packing properties, and intermolecular interactions of two structural polymorphs of N-palmitoylethanolamine (NPEA) have been determined by single-crystal X-ray diffraction. Polymorphs alpha and beta crystallized in monoclinic space group P2(1)/c and orthorhombic space group Pbca, respectively. In both polymorphs, NPEA molecules are organized in a tail-to-tail manner, resembling a bilayer membrane. Although the molecular packing in polymorph alpha is similar to that in N-myristoylethanolamine and N-stearoylethanolamine, polymorph beta is a new form. The acyl chains in both polymorphs are tilted by approximately 35 degrees with respect to the bilayer normal, with their hydrocarbon moieties packed in an orthorhombic subcell. In both structures, the hydroxy group of NPEA forms two hydrogen bonds with the hydroxy groups of molecules in the opposite leaflet, resulting in extended, zig-zag type H-bonded networks along the b-axis in polymorph alpha and along the a-axis in polymorph beta. Additionally, the amide N-H and carbonyl groups of adjacent molecules are involved in N-H...O hydrogen bonds that connect adjacent molecules along the b-axis and a-axis, respectively, in alpha and beta. Whereas in polymorph alpha the L-shaped NPEA molecules in opposite layers are arranged to yield a Z-like organization, in polymorph beta one of the two NPEA molecules is rotated 180 degrees , leading to a W-like arrangement. Lattice energy calculations indicate that polymorph alpha is more stable than polymorph beta by approximately 2.65 kcal/mol.     

Expression of hepatitis B surface antigen in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> utilizing glyceraldeyhyde-3-phosphate dehydrogenase promoter of <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An expression vector constructed from genes of Pichia pastoris was applied for heterologous gene expression in Saccharomyces cerevisiae. Recombinant hepatitis B surface antigen was synthesized by cloning hepatitis B virus ‘S’ gene under the control of glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter of Pichia pastoris in Saccharomyces cerevisiae. Hepatitis B surface antigen was constitutively expressed, was stable and exhibited ∼20–22 nm particle formation. Stability and absence of toxicity to the host with the expression vector indicates the expression system can be applied for large-scale production.     

Divergence of larval resource acquisition for water conservation and starvation resistance in Drosophila melanogaster

Laboratory selection experiments have evidenced storage of energy metabolites in adult flies of desiccation and starvation resistant strains of D. melanogaster but resource acquisition during larval stages has received lesser attention. For wild populations of D. melanogaster, it is not clear whether larvae acquire similar or different energy metabolites for desiccation and starvation resistance. We tested the hypothesis whether larval acquisition of energy metabolites is consistent with divergence of desiccation and starvation resistance in darker and lighter isofemale lines of D. melanogaster. Our results are interesting in several respects. First, we found contrasting patterns of larval resource acquisition, i.e., accumulation of higher carbohydrates during 3rd instar larval stage of darker flies versus higher levels of triglycerides in 1st and 2nd larval instars of lighter flies. Second, 3rd instar larvae of darker flies showed ~40?h longer duration of development at 21°C; and greater accumulation of carbohydrates (trehalose and glycogen) in fed larvae as compared with larvae non-fed after 150?h of egg laying. Third, darker isofemale lines have shown significant increase in total water content (18%); hemolymph (86%) and dehydration tolerance (11%) as compared to lighter isofemale lines. Loss of hemolymph water under desiccation stress until death was significantly higher in darker as compared to lighter isofemale lines but tissue water loss was similar. Fourth, for larvae of darker flies, about 65% energy content is contributed by carbohydrates for conferring greater desiccation resistance while the larvae of lighter flies acquire 2/3 energy from lipids for sustaining starvation resistance; and such energy differences persist in the newly eclosed flies. Thus, larval stages of wild-caught darker and lighter flies have evolved independent physiological processes for the accumulation of energy metabolites to cope with desiccation or starvation stress.     

Multiparameter assessments to determine the effects of sugars and antimicrobials on a polymicrobial oral biofilm

Clinical studies indicate relationships between dental plaque, a naturally formed biofilm, and oral diseases. The crucial role of nonmicrobial biofilm constituents in maintaining biofilm structure and biofilm-specific attributes, such as resistance to shear and viscoelasticity, is increasingly recognized. Concurrent analyses of the diverse nonmicrobial biofilm components for multiparameter assessments formed the focus of this investigation. Comparable numbers of Actinomyces viscosus, Streptococcus sanguinis, Streptococcus mutans, Neisseria subflava, and Actinobacillus actinomycetemcomitans cells were seeded into multiple wells of 96-well polystyrene plates for biofilm formation. Quantitative fluorescence and confocal laser scanning microscopy (CLSM) examined the influences of dietary sugars, incubation conditions, ingredients in oral hygiene formulations, and antibiotics on biofilm components. Biofilm extracellular polymeric substances (EPS) were examined with an optimized mixture of fluorescent lectins, with biofilm proteins, lipids, and nucleic acids detected with specific fluorescent stains. Anaerobic incubation of biofilms resulted in significantly more biofilm EPS and extractable carbohydrates than those formed under aerobic conditions (P < 0.05). Sucrose significantly enhanced biofilm EPS in comparison to fructose, galactose, glucose, and lactose (P < 0.05). CLSM demonstrated thicker biofilms under sucrose-replete conditions, along with significant increases in biofilm EPS, proteins, lipids, and nucleic acids, than under conditions of sucrose deficiency (P < 0.05). Agents in oral hygiene formulations (chlorhexidine, ethanol, and sodium lauryl sulfate), a mucolytic agent (N-acetyl-L-cysteine), and antibiotics with different modes of action (amoxicillin, doxycycline, erythromycin, metronidazole, and vancomycin) inhibited biofilm components (P < 0.05). Multiparameter analysis indicated a dose-dependent inhibition of biofilm EPS and protein by chlorhexidine and sodium lauryl sulfate, along with distinctive inhibitory patterns for subinhibitory concentrations of antibiotics. Collectively, these results highlight multiparameter assessments as a broad platform for simultaneous assessment of diverse biofilm components.