A novel approach in the assessment of polymeric film formation and film adhesion on different pharmaceutical solid substrates

The purpose of this study was to evaluate the nature of film formation on tablets with different compositions, using confocal laser scanning microscopy (CLSM), and to measure film adhesion via the application of a novel “magnet probe test”. Three excipients, microcrystalline cellulose (MCC), spray-dried lactose monohydrate, and dibasic calcium phosphate dihydrate, were individually blended with 0.5% magnesium stearate, as a lubricant, and 2.5% tetracycline HCl, as a fluorescent marker, and were compressed using a Carver press. Tablets were coated with a solution consisting of 7% hydroxypropyl methylcellulose (HPMC) phthalate (HP-55), and 0.5% cetyl alcohl in acetone and isopropanol (11:9). The nature of polymer interaction with the tablets and coating was evaluated using CLSM and a designed magnet probe test. CLSM images clearly showed coating efficiency, thickness, and uniformity of film formation, and the extent of drug migration into the film at the coating interfaces of tablets. Among the excipients, MCC demonstrated the best interface for both film formation and uniformity in thickness relative to lactose monohydrate and dibasic calcium phosphate dihydrate. The detachment force of the coating layers from the tablet surfaces, as measured with the developed magnet probe test, was in the order of MCC>lactose monohydrate>dibasic calcium phosphate dihydrate. It was also shown that the designed magnet probe test provides reliable and reproducible results when used for measurement of film adhesion and bonding strength.     

Changes in elemental concentrations in LNCaP cells are associated with a protective effect of neuropeptides on etoposide-induced apoptosis

One of the mechanisms that has been put forward for the development of the androgen-resistant status is neuroendocrine differentiation. Neuroendocrine cells secrete neuropeptides that may represent one of the possible molecular bases by which hormone-dependent prostate cancer cells could escape treatment. LNCaP prostate cancer cells were treated with either etoposide or neuropeptides. Morphological changes related to apoptosis and cell viability were assessed. Changes in intracellular ion content were quantitatively analyzed by electron probe X-ray microanalysis. Etoposide treatment consistently induces a decrease in K and an increase in Na, which are inhibited by bombesin or calcitonin. The Na/K ratio increased markedly after exposure to etoposide, and both bombesin and calcitonin blocked this increase. Etoposide also caused changes in the intracellular P and S concentrations that to a large extent could be blocked by neuropeptides. These results support the hypothesis that neuropeptides confer anti-apoptotic capabilities onto non-neuroendocrine cells in close proximity to neuroendocrine cells.     

A molecular marker diagnostic of a specific isolate of an arbuscular mycorrhizal fungus,Gigaspora margarita

To investigate the auto-ecology of a strain of Gigaspora margarita in a commercial inoculum, we found a pair of PCR primers amplifying a sequence of 235 bp diagnostic of the isolate. We designed an oligonucleotide probe based on the DNA sequence. The combination of PCR and the probing successfully detected the diagnostic sequence from both DNA preparations of single spores and colonized roots. This protocol enabled us to distinguish the isolate among several isolates from Japan, Nepal and the USA.     

Assembly of light-harvesting bacteriochlorophyll in a model transmembrane helix in its natural environment

The transmembrane, bacteriochlorophyll-binding region of a bacterial light-harvesting complex, (LH2-alpha from the photosynthetic bacterium Rhodobacter sphaeroides) was redesigned and overexpressed in a mutant of Rb. sphaeroides lacking LH2. Bacteriochlorophyll served as internal probe for the fitness of this new region for the assembly and energy transfer function of the LH2 complex. The ability to absorb and transfer light energy is practically undisturbed by the exchange of the transmembrane segment, valine -7 to threonine +6, of LH2-alpha with a 14 residue Ala-Leu sequence. This stretch makes up the residues of the transmembrane helix that are in close contact (< or =4.5 A) with the bacteriochlorophyll molecules that are coordinated through His of both the alpha and beta-subunits. In this Ala-Leu stretch, neither alpha-His0, which binds the bacteriochlorophyll, nor the adjacent alpha-Ile-1, were replaced. Novel LH2 complexes composed of LH2-alpha with a model transmembrane sequence and a normal LH2-beta are assembled in vivo into a complex, the biochemical and spectroscopic properties of which closely resemble the native one. In contrast, the additional insertion of four residues just outside the C-terminal end of the model transmembrane helix leads to complete loss of functional antenna complex. The results suggest that light energy can be harvested and transferred efficiently by bacteriochlorophyll molecules attached to only few key residues distributed over the polypeptide, while residues at the bacteriochlorophyll-helix interface seem to be largely dispensable for the functional assembly of this membrane protein complex. This novel antenna with a simplified transmembrane domain and a built-in probe for assembly and function provides a powerful model system for investigation of the factors that contribute to the assembly of chromophores in membrane-embedded proteins.     

Biological properties of a new fluorescent biphalin fragment analogue

Previous studies of structure-activity of biphalin defined fragments which expressed the full biological potency of the parent compound. The most simple fragment was Tyr-D-Ala-Gly-Phe-NH-NH<--X, where X=Phe, but it also could be other hydrophobic amino acids. This paper presents data that replacement of the phenylalanine with a dansyl (X=DNS) groups gives an analogue (AA2016) that fully preserves the high affinity of the initial analogue for both mu and delta opioid receptors. In the tail flick test in rats, intrathecal injection of the compound produces strong antinociception, comparable to the parent biphalin. Because AA2016 contains a strong fluorescent group, it can be a very useful tool for prospective studies in vivo, including biological barrier permeability, tissue distribution, metabolism and receptor-ligand complex formation.     

Terbium(III) fluorescence probe studies on metal ion-binding sites in anticoagulation factor I from Agkistrodon acutus venom

Anticoagulation factor I (ACF I) isolated from the venom of Agkistrodon acutus is an activated coagulation factor X-binding protein with marked anticoagulant activity. Present studies show that holo-ACF I assumes a compactly folded structure in the range of pH 5–6, in which the most interior Trp residues and quenchers are adjacent. Tb³⁺ ions can completely replace both Ca²⁺ ions in holo-ACF I, as determined by equilibrium dialysis. Although the two Tb³⁺ ions in Tb³⁺-ACF I have slightly different luminescence efficiencies, both have similar quenching effects on the intrinsic fluorescence, suggesting that probably there are same numbers of Trp residues close to both Tb³⁺-binding sites. Two Tb³⁺-binding sites with similar apparent Tb³⁺ association constant values, (1.69 ± 0.02) × 10⁷ M^–1 and (1.42 ± 0.01) × 10⁷ M^–1, respectively, were further identified through Tb³⁺ fluorescence titration. In addition, it has been confirmed from the titration of holo-ACF I and Tb³⁺-ACF I with NBS that only interior Trp residues are involved in the energy transfer to Tb³⁺ ions and that all accessible Trp residues located in the surface of holo-ACF I have similar affinity to NBS, while those located in the surface of Tb³⁺-ACF I have two different kinds of affinity to NBS, which strongly suggests a conformational change of holo-ACF I upon substitution of Tb³⁺ for Ca²⁺. The results show that although the Tb³⁺-altered conformation of ACF I cannot support the binding of Tb³⁺-ACF I with FXa, determined by nondenaturing PAGE, Tb³⁺ ions are effective and useful fluorescence probes to analyze the structures and properties of Ca²⁺-binding sites in ACF I.     

Rapid changes in levels of mineral nutrients in root-tip cells following short-term exposure to aluminium

We investigated the rapid modification of plasma membrane and changes in mineral nutrients in root-tip cells of Al-tolerant rice and Al-sensitive barley following short-term exposure to Al (20 M Al, 1 h). The plasma membrane of the barley cells was significantly permeabilized when re-elongated in an Al-free Ca solution following a 1-h pre-treatment with Al, while that of rice cells was not affected at all. The elemental distribution and concentration in a 2-mm portion of the root apex were determined by electron probe X-ray microanalysis. Al was localized primarily to the epidermis and outer cortex cells in both species, and was much more abundant in barley than in rice. Al increased and decreased remarkably the intracellular K concentration in whole root-tip cells of rice and barley, respectively. In barley, the decrease in the concentration of Ca coincided with the accumulation of Al. Conversely, the intracellular concentration of P in the surface layers of root-tip cells increased with the accumulation of Al. The distribution and concentration of Ca and P in rice did not change after 1-h treatment with Al. These results suggest that the rapid modification of the plasma membrane of root-tip cells induced by Al affects the nutritional homeostasis in the cells.     

Molecular phylogenetic profiling of prokaryotic communities in guts of termites with different feeding habits

Termites are an important group of terrestrial insects that harbor an abundant gut microbiota, many of which contribute to digestion, termite nutrition and gas (CH(4), CO(2) and H(2)) emission. With 2200 described species, termites also provide a good model to study relationships between host diet and gut microbial community structure and function. We examined the relationship between diet and gut prokaryotic community profiles in 24 taxonomically and nutritionally diverse species of termites by using nucleic acid probes targeting 16S-like ribosomal RNAs. The relative abundance of domain-specific 16S-like rRNAs recovered from gut extracts varied considerably (ranges: Archaea (0-3%); Bacteria (15-118%)). Although Bacteria were always detectable and the most abundant, differences in domain-level profiles were correlated with termite diet, as evidenced by higher relative abundances of Archaea in guts of soil-feeding termites, compared to those of wood-feeding species in the same family. The oligonucleotide probes also readily distinguished gut communities of wood-feeding taxa in the family Termitidae (higher termites) from those of other wood-feeding termite families (lower termites). The relative abundances of 16S-like archaeal rRNA in guts were positively correlated with rates of methane emission by live termites, and were consistent with previous work linking high relative rates of methanogenesis with the soil (humus)-feeding habit. Probes for methanogenic Archaea detected members of only two families (Methanobacteriaceae and Methanosarcinaceae) in termite guts, and these typically accounted for 60% of the all archaeal probe signal. In four species of termites, Methanosarcinaceae were dominant, a novel observation for animal gut microbial communities, but no clear relationship was apparent between methanogen family profiles and termite diet or taxonomy.     

Environmental factors influencing the distribution of rRNA from Verrucomicrobia in soil

The Verrucomicrobia constitute a newly discovered division of the Bacteria identified as a numerically abundant component of soil microbial communities in numerous sites around the world. The relative abundance of rRNA from Verrucomicrobia was investigated in the soil to examine the influence of specific environmental factors on the distribution of Verrucomicrobia and to better understand the distribution of this group in terrestrial ecosystems. The abundance of the verrucomicrobial rRNA was determined by using a novel oligonucleotide probe that is specific for verrucomicrobial 16S rRNA. The abundance of verrucomicrobial 16S rRNA in soil microbial communities was determined in relation to plant community composition and soil management history over a period of 2 years. Additional samples were analyzed to determine if verrucomicrobial rRNA relative abundance changes in relation to either soil depth or soil moisture content. The Verrucomicrobia composed 1.9+/-0.2% of the microbial community rRNA present in the 85 soil samples examined. The distribution of verrucomicrobial rRNA in the soil reveals that Verrucomicrobia are significantly affected by environmental characteristics that change in relation to time, soil history, and soil depth, and reveals that a statistically significant amount of the variation in verrucomicrobial rRNA abundance can be explained by changes in soil moisture content.     

New broad-host-range promoter probe vectors based on the plasmid RK2 replicon

Broad-host-range plasmid RK2-based promoter probe vectors with a known nucleotide sequence were constructed. In the absence of an upstream promoter, the expression of two tested reporter genes (luc and lacZ) in Escherichia coli was virtually zero, while insertion of the Ptrc promoter resulted in strong inducer-dependent expression. The lacZ-based vectors were mobilized into Pseudomonas fluorescens ST, Pseudomonas putida KT2442, Sphingomonas spp. and Burkholderia spp. LB400, and expression analyses indicated that the properties observed in E. coli are maintained across the species barriers. In addition, the previously established knowledge of RK2 molecular biology allows easy manipulations of features such as plasmid copy number, further extending the application potential of the vectors.