The magnum-isthmus junction of the fowl oviduct participates in the formation of the avian-type shell membrane

Avian eggs possess a shell membrane in the shape of an asymmetrical ellipsoid and with a limiting membrane that is a smooth layer of homogeneous, dense materials. We describe the role of the magnum-isthmus junction (MIJ) of the oviduct in the formation of the avian-type shell membrane in the domestic fowl Gallus domesticus. The narrow width of the lumen at the MIJ indirectly participates in the determination of the asymmetrical ellipsoid shape of eggs that are encased by the egg-white layer and subsequently by the peri-albumen layer (PL) and the shell membrane. The PL reacts with Alcian blue and exists between the egg white and the limiting membrane. It is added to the ovulating egg at the MIJ and covers the outermost surface of the egg-white layer. The function of the PL is to provide a smooth surface by covering the irregular surface of the egg-white layer. The materials of the PL consist of an Alcian blue-positive polysaccharide (or glycoprotein) of 240 kDa and five proteins of 135, 116, 72, 49, and 46 kDa. The isolated materials have an affinity to bind with the egg-white mass. An antiserum against quail PL materials stains the domestic fowl PL and secretory cells of the luminal epithelium at the MIJ, and cross-reacts with the molecules of 240, 135, and 116 kDa.     

Short synthesis of 16beta-hydroxy-5alpha-cholestane-3,6-dione a novel cytotoxic marine oxysterol

The first and short synthesis of 16beta-hydroxy-5alpha-cholestane-3,6-dione 1 a metabolite from marine algae, has been achieved in six steps from readily available diosgenin 5. Selective deoxygenation of primary alcohol of triol 6 has been accomplished in one step using Et(3)SiH and catalytic amount of B(C(6)F(5))(3) to produce compound 9 in high yield. Oxidation of 11 with PCC, allowed the introduction of 3,6-ene-dione functionality, and further catalytic hydrogenation and deprotection furnished the 3,6-diketo steroid 1.     

The effect of paragigantocellularis lateralis lesion on conditioned place preference (CPP) in presence or absence of alpha2 adrenergic agonist (clonidine) in male rats

The nucleus paragigantocellularis lateralis (LPGi) is located in the rostral ventrolateral medulla (RVLM), a brain stem region that regulates homeostatic functions such as blood pressure and cardiovascular reflexes, respiration, pain and opiate withdrawal syndrome. LPGi has many anatomical relationships with important nuclei such as arcute nucleus, caudal raphe nucleus, periaqueductal gray (PAG), locus coeruleus (LC), and dentate. In this study we have examined the role of LPGi in the conditioned place preference (CPP) induced by morphine in the presence and absence of clonidine in the rat. We used 49 male N-MRI rats which were divided into 7 groups randomly: 1: Control, 2: Control+saline, 3: sham control, 4: lesion, 5: lesion +0.02 mg/kg clonidine, 6: lesion +0.2 mg/kg clonidine, 7: lesion +2 mg/kg clonidine. Animals were anaesthetized with ketamine (110 mg/kg) and rampune (Xylazine) (3 mg/kg) mixture. In the process of surgery LPGi nucleus has been destroyed bilaterally by DC electrical current (1 mA, 6 second), with stainless steel electrode placed in stereotaxic coordinates of (AP = 11.8, Lat +/- 1.86 and Depth = 10.5). After the recovery period, they were treated with clonidine one hour before the application of Hand's method to induce CPP. We have not found any significant differences between the results of control, control+saline and sham groups in the CPP test but there is a significant increase in the CPP time between sham and LPGi lesion+saline groups (P < 0.019). Clonidine at different doses (0.02, 0.2 and 2 mg/kg) have decreased CPP time in LPGi lesioned group in comparison with lesioned+saline group as well (p < 0.002). In this study we have also demonstrated that clonidine has not any effects on the CPP time in the intact animals. Our results indicate that LPGi lesion induces CPP. It seems that LPGi is involved in drug reinforcements and also LPGi lesion induces sensitivity to alpha2 adrenergic agonist.     

Protein refolding assisted by molecular tube based α-cyclodextrin as an artificial chaperone

In this study, we evaluated, for the first time, the application of molecular tube based alpha-cyclodextrin for improving the refolding yields of two different enzymes: carbonic anhydrase and alkaline phosphatase. Our results indicate that under the optimal developed refolding environments, the denatured carbonic anhydrase and alkaline phosphatase were refolded with a yield of 51 and 61% using 15 and 5 mg/ml of the molecular tube, respectively. Regardless of lower refolding yields compared with liquid-phase artificial chaperone assisted approach, the new technique (solid-phase artificial chaperone assisted refolding) benefits from easier and faster separation of the refolded product from the refolding environment, recycling of the stripping agent, and finally, significantly less environmental effect at the industrial levels. However, further improvements in solid-phase artificial chaperone assisted technique are needed either through synthesizing better stripping agents or by optimizing and defining better refolding environments.     

Ultrastructural comparison of developing mouse embryonic stem cell- and in vivo-derived cardiomyocytes

Embryonic stem cells (ESCs) are expected to become a powerful tool for future regenerative medicine and developmental biology due to their capacity for self-renewal and pluripotency. The present study involves characterization and particularly, the ultrastructure of ESC-derived cardiomyocytes (ESC-CMs). Spontaneously differentiated murine (C57BL/6) ESC-CMs were cultured for 21 days. At different stages, growth characteristics of the CMs were assessed by immunocytochemistry, RT-PCR, transmission electron microscopy, and by addition of chronotropic drugs. EB-derived spontaneously beating cells expressed markers characteristic of CMs including alpha-actinin, desmin, troponin I, sarcomeric myosin heavy chain (MHC), pan-cadherin, connexin 43, cardiac alpha-MHC, cardiac beta-MHC, atrial natriuretic factor (ANF), and myosin light chain isoform-2V (MLC-2V) and responded to drugs in a maturation- and dose-dependent manner. At the ultrasructural level, maturation proceeded with increasing time in culture. In 7+21 days CMs, all sarcomeric components, such as Z-discs, A-, I- and H-bands as well as M-lines, T-tubules, intercalated discs, and the sarcoplasmic reticulum were present. Our data suggest that ESCs can differentiate into functional mature CMs in vitro. Furthermore, ESC-CMs may provide an ideal model for the study of cardiomyocytic development and may be useful for cell therapy of various cardiac diseases.     

In silico pharmacogenetics of warfarin metabolism

Pharmacogenetic approaches can be instrumental for predicting individual differences in response to a therapeutic intervention. Here we used a recently developed murine haplotype-based computational method to identify a genetic factor regulating the metabolism of warfarin, a commonly prescribed anticoagulant with a narrow therapeutic index and a large variation in individual dosing. After quantification of warfarin and nine of its metabolites in plasma from 13 inbred mouse strains, we correlated strain-specific differences in 7-hydroxywarfarin accumulation with genetic variation within a chromosomal region encoding cytochrome P450 2C (Cyp2c) enzymes. This computational prediction was experimentally confirmed by showing that the rate-limiting step in biotransformation of warfarin to its 7-hydroxylated metabolite was inhibited by tolbutamide, a Cyp2c isoform-specific substrate, and that this transformation was mediated by expressed recombinant Cyp2c29. We show that genetic variants responsible for interindividual pharmacokinetic differences in drug metabolism can be identified by computational genetic analysis in mice.     

A three-level problem-centric strategy for selecting NMR precursor labeling and analytes

We have developed a sequential set of computational screens that may prove useful for evaluating analyte sets for their ability to accurately report on metabolic fluxes. The methodology is problem-centric in that the screens are used in the context of a particular metabolic engineering problem. That is, flux bounds and alternative flux routings are first identified for a particular problem, and then the information is used to inform the design of nuclear magnetic resonance (NMR) experiments. After obtaining the flux bounds via MILP, analytes are first screened for whether the predicted NMR spectra associated with various analytes can differentiate between different extreme point (or linear combinations of extreme point) flux solutions. The second screen entails determining whether the analytes provide unique flux values or multiple flux solutions. Finally, the economics associated with using different analytes is considered in order to further refine the analyte selection process in terms of an overall utility index, where the index summarizes the cost-benefit attributes by quantifying benefit (contrast power) per cost (e.g., NMR instrument time required). We also demonstrate the use of an alternative strategy, the Analytical Hierarchy Process, for ranking analytes based on the individual experimentalist's-generated weights assigned for the relative value of flux scenario contrast, unique inversion of NMR data to fluxes, etc.     

An experiment on comb orientation by honey bees (Hymenoptera: Apidae) in traditional hives

The orientation of combs in traditional beehives is extremely important for obtaining a marketable honey product. However, the factors that could determine comb orientation in traditional hives and the possibilities of inducing honey bees, Apis mellifera (L.), to construct more desirable combs have not been investigated. The goal of this experiment was to determine whether guide marks in traditional hives can induce bees to build combs of a desired orientation. Thirty-two traditional hives of uniform dimensions were used in the experiment. In 24 hives, ridges were formed on the inner surfaces of the hives with fermented mud to obtain different orientations, circular, horizontal, and spiral, with eight replicates of each treatment. In the remaining eight control hives, the inner surface was left smooth. Thirty-two well-established honey bee colonies from other traditional hives were transferred to the prepared hives. The colonies were randomly assigned to the four treatment groups. The manner of comb construction in the donor and experimental hives was recorded. The results showed that 22 (91.66%) of the 24 colonies in the treated groups built combs along the ridges provided, whereas only 2 (8.33%) did not. Comb orientation was strongly associated with the type of guide marks provided. Moreover, of the 18 colonies that randomly fell to patterns different from those of their previous nests, 17 (94.4%) followed the guide marks provided, irrespective of the comb orientation type in their previous nest. Thus, comb orientation appears to be governed by the inner surface pattern of the nest cavity. The results suggest that even in fixed-comb hives, honey bees can be guided to build combs with orientations suitable to honey harvesting, without affecting the colonies.     

Cholesterol superlattice modulates CA4P release from liposomes and CA4P cytotoxicity on mammary cancer cells

Liposomal drugs are a useful alternative to conventional drugs and hold great promise for targeted delivery in the treatment of many diseases. Most of the liposomal drugs on the market or under clinical trials include cholesterol as a membrane stabilizing agent. Here, we used liposomal CA4P, an antivascular drug, to demonstrate that cholesterol content can actually modulate the release and cytotoxicity of liposomal drugs in a delicate and predictable manner. We found that both the rate of the CA4P release from the interior aqueous compartment of the liposomes to the bulk aqueous phase and the extent of the drug's cytotoxicity undergo a biphasic variation, as large as 50%, with liposomal cholesterol content at the theoretically predicted C(r), e.g., 22.0, 22.2, 25.0, 33.3, 40.0, and 50.0 mol % cholesterol for maximal superlattice formation. It appears that at C(r), CA4P can be released from the liposomes more readily than at non-C(r), probably due to the increased domain boundaries between superlattice and nonsuperlattice regions, which consequently results in increased cytotoxicity. The idea that the increased domain boundaries at C(r) would facilitate the escape of molecules from membranes was further supported by the data of dehydroergosterol transfer from liposomes to MβCD. These results together show that the functional importance of sterol superlattice formation in liposomes can be propagated to distal targeted cells and reveal a new, to our knowledge, mechanism for how sterol content and membrane lateral organization can control the release of entrapped or embedded molecules in membranes.