Large-Scale Preparation of Recombinant Ovine Prolactin and Determination of Its in Vitro and in Vivo Activity

Recombinant bovine Ala-prolactin (PRL) (GenBank Accession No. V00112) in prokaryotic expression plasmid pMON3401 was mutated using a mutagenesis kit, to prepare plasmid encoding ovine PRL (oPRL) (GenBank Accession No. M27057) Escherichia coli cells transformed with this latter plasmid overexpressed large amounts of oPRL upon induction with nalidixic acid. The expressed protein, found in inclusion bodies, was refolded and purified to homogeneity on a Q-Sepharose column, yielding an electrophoretically pure fraction composed of over 98% monomeric protein of the expected molecular mass of 23 kDa. The biological activity of the recombinant oPRL after proper renaturation was evidenced in vitro by its ability to stimulate proliferation of rat lymphoma Nb₂ cells possessing PRL receptors, to stimulate luciferase activity in HEK 293 cells transiently transfected with oPRL receptors, and to induce progesterone secretion in primary cultures of luteal cells obtained from midpregnant ewes. In contrast to ovine growth hormone or ovine placental lactogen, recombinant oPRL had no galactopoietic effect in lactating ewes.     

Woody vegetation composition and diversity in woodlands inside and outside a Forest Reserve in Jos,Nigeria

Protected areas such as forest reserves are often assumed to be best ways to conserve biodiversity and maintain intact ecosystems. We examined woody plant composition and diversity in the gallery forest and savannah woodland habitats of Amurum Forest Reserve and areas immediately surrounding it in Jos, Nigeria. A total of 100 10 × 10 m sample plots were established inside and outside the reserve. All woody plants ≥1 cm diameter at breast height (dbh) were identified and measured. A total of 7,564 individual plants categorized as 134 species from 44 families were recorded. Overall species diversity was significantly higher in the Forest Reserve than outside the reserve, although more species were encountered outside the reserve. Our findings suggest that, protected areas and the areas surrounding them are important for the conservation of biodiversity as the areas outside protected areas also contain species of conservation value. The continuous degrading areas outside protected areas isolates them and poses a serious threat to the long‐term viability of wildlife populations, so it is important that biodiversity in protected areas and their surrounding areas be conserved.     

Integrating Ethnography and Hunting Sustainability Modeling for Primate Conservation in an Indigenous Reserve in Guyana

Indigenous reserves are increasingly common throughout the tropical world. This is particularly true in Amazonia, where they make up >50% of protected land area. While these reserves offer tremendous opportunities for conservation, hunting represents a considerable threat to primate populations. As eliminating hunting may not be feasible, conservationists must work collaboratively with indigenous groups to promote sustainable management. This requires an understanding of the sociocultural drivers of hunting, quantitatively assessing sustainability, and developing co-management strategies that are commensurable with indigenous ontologies. In this article, we integrate ethnography with sustainability modeling to assess the importance of primate hunting to the livelihoods and culture of indigenous Waiwai in the Konashen Community-Owned Conservation Area, Guyana and to simultaneously promote sustainable co-management. We collected quantitative data on Waiwai harvesting through hunter self-monitoring and used semistructured interviews, unstructured interviews, and participant observation to understand the cultural importance of hunting to Waiwai society. We incorporated these data into spatially explicit biodemographic models to assess sustainability for four primate species. Primates, particularly spider monkeys (Ateles pansicus), were among the most important Waiwai prey and primate hunting played an important role in the construction of both individual and collective Waiwai identity. Our biodemographic models indicated that hunting will cause relatively little depletion for most primates in 20 years, although spider monkeys are predicted to disappear from a majority of the Waiwai catchment area. We argue that successful co-management of hunting in indigenous reserves requires truly integrative approaches that combine quantitative sustainability assessments with detailed concurrent ethnographic research.     

Characterization of the fluorescence of the protamine thynnine and studies of binding to double-stranded DNA

The protamine thynnine is an arginine-rich protein approximately 30 amino acids long with a tyrosine in the middle of its sequence. Its fluorescence decay kinetics can be described by a biexponential function with lifetimes of 0.52 and 2.1 ns, with almost equal preexponential factors. The fluorescence quencher CsCl does not affect the short lifetime but shifts the equilibrium between the long and short lifetime toward the short one and reduces the long lifetime. In nature, thynnine is found complexed with chromosomal DNA. In vitro complexes of thynnine with double-stranded (ds) DNA are stable at physiologic ionic strength but dissociate at high NaCl concentration. This dissociation can be monitored by steady-state fluorescence. From the salt concentration dependence of the dissociation of the complex of thynnine with ds-DNA 145 bp long, it can be concluded that only 4 of 21 possible full electrostatic bonds are involved in thynnine-DNA binding. In addition, the binding constant at 1M NaCl is of the order of 10⁶, indicating a strong nonelectrostatic component in arginine-DNA binding.     

MzrA: a novel modulator of the EnvZ/OmpR two-component regulon

Analysis of suppressors that alleviate the acute envelope stress phenotype of a Δ bamB Δ degP strain of Escherichia coli identified a novel protein MzrA and pleiotropic envZ mutations. Genetic evidence shows that overexpression of MzrA – formerly known as YqjB and EcfM – modulates the activity of EnvZ/OmpR similarly to pleiotropic EnvZ mutants and alter porin expression. However, porin expression in strains devoid of MzrA or overexpressing it is still sensitive to medium osmolarity, pH and procaine, all of which modulate EnvZ/OmpR activities. Thus, MzrA appears to alter the output of the EnvZ/OmpR system but not its ability to receive and respond to various environmental signals. Localization and topology experiments indicate that MzrA is a type II membrane protein, with its N-terminus exposed in the cytoplasm and C-terminus in the periplasm. Bacterial two-hybrid experiments determined that MzrA specifically interacts with EnvZ but not with OmpR or the related membrane sensor kinase, CpxA. This and additional genetic and biochemical evidence suggest that the interaction of MzrA with EnvZ would either enhance EnvZ's kinase activity or reduce its phosphatase activity, thus elevating the steady state levels of OmpR∼P. Furthermore, our data show that MzrA links the two-component envelope stress response regulators, CpxA/CpxR and EnvZ/OmpR.     

Modulation of functionally significant conformational equilibria in adenylate kinase by high concentrations of trimethylamine oxide attributed to volume exclusion

The effect of an inert small molecule osmolyte, trimethyl amine N-oxide (TMAO), upon the conformational equilibria of Escherichia coli adenylate kinase was studied using time-resolved FRET. The relative populations of open and closed clefts between the LID and the CORE domains were measured as functions of the concentrations of the substrate ATP over the concentration range 0–18 mM and TMAO over the concentration range 0–4 M. A model was constructed according to which the enzyme exists in equilibrium among four conformational states, corresponding to combinations of open and closed conformations of the LID-CORE and AMP-CORE clefts. ATP is assumed to bind only to those conformations with the closed LID-CORE cleft, and TMAO is assumed to be differentially excluded as a hard spherical particle from each of the four conformations in accordance with calculations based upon x-ray crystallographic structures. This model was found to describe quantitatively the dependence of the fraction of the closed LID-CORE cleft upon the concentrations of both ATP and TMAO over the entire range of concentrations with just five undetermined parameters.     

L-Myo-inositol 1-phosphate synthase in the aquatic fern Azolla filiculoides.

L-Myo-inositol 1-phosphate synthase (INPS EC 5.5.1.4) catalyzes the conversion of D-glucose 6-phosphate to L-myo-inositol 1-phosphate. INPS is a key enzyme involved in the biosynthesis of phytate which is a common form of stored phosphates in higher plants. The present study monitored the increase of INPS expression in Azolla filiculoides resulting from exposure to inorganic phosphates, metals and salt stress. The expression of INPS was significantly higher in Azolla plants that were grown in rich mineral growth medium than those maintained on nutritional growth medium. The expression of INPS protein and corresponding mRNA increased in plants cultured in minimal nutritional growth medium when phosphate or Zn2+, Cd2+ and NaCl were added to the growth medium. When employing rich mineral growth medium, INPS protein content increased with the addition of Zn2+, but decreased in the presence of Cd2+ and NaCl. These results indicated that accumulation of phytate in Azolla is a result of the intensified expression of INPS protein and mRNA, and its regulation may be primarily derived by the uptake of inorganic phosphate, and Zn2+, Cd2+ or NaCl.     

Characterization and Evaluation of 5-Fluorouracil-Loaded Solid Lipid Nanoparticles Prepared via a Temperature-Modulated Solidification Technique

The aim of this research was to advance solid lipid nanoparticle (SLN) preparation methodology by preparing glyceryl monostearate (GMS) nanoparticles using a temperature-modulated solidification process. The technique was reproducible and prepared nanoparticles without the need of organic solvents. An anticancer agent, 5-fluorouracil (5-FU), was incorporated in the SLNs. The SLNs were characterized by particle size analysis, zeta potential analysis, differential scanning calorimetry (DSC), infrared spectroscopy, atomic force microscopy (AFM), transmission electron microscopy (TEM), drug encapsulation efficiency, in vitro drug release, and in vitro cell viability studies. Particle size of the SLN dispersion was below 100 nm, and that of redispersed lyophilizates was ~500 nm. DSC and infrared spectroscopy suggested that the degree of crystallinity did not decrease appreciably when compared to GMS. TEM and AFM images showed well-defined spherical to oval particles. The drug encapsulation efficiency was found to be approximately 46%. In vitro drug release studies showed that 80% of the encapsulated drug was released within 1 h. In vitro cell cultures were biocompatible with blank SLNs but demonstrated concentration-dependent changes in cell viability to 5-FU-loaded SLNs. The 5-FU-loaded SLNs can potentially be utilized in an anticancer drug delivery system.KEY WORDS: atomic force microscopy, calorimetry (DSC), FTIR, particle size, solid lipid nanoparticles