6-Phosphofructo-2-kinase and D-fructose-2,6-bisphosphatase activities in mung bean seedlings

6-Phosphofructo-2-kinase (ATP: D-fructose-6-phosphate-2-phosphotransferase) and D-fructose-2,6-bisphosphatase activities have been found in extracts prepared from etiolated mung bean seedlings. The activity of 6-phosphofructo-2-kinase exhibits a sigmoidal shape in response to changes in concentrations of both substrates, D-fructose 6-phosphate and ATP (S_0.5 values of 1.8 and 1.2 mM, respectively). Inorganic orthophosphate (P_i) has a strong stimulating effect on the 2-kinase activity (A_0.5 at about 2 mM), moderately increasing the V_max and modifying the response into hyperbolic curves with K_m values of 0.4 and 0.2 mM for fructose 6-phosphate and ATP, respectively. 3-Phosphoglycerate (I_0.5 about 0.15 mM) partially inhibited the kinase activity by counteracting the P_i activation. In contrast, the activity of D-fructose-2,6-bisphosphatase (K_m 0.38 mM) is strongly inhibited by P_i (I_0.5 0.8 mM) lowering its affinity to fructose-2,6-P₂ (K_m 1.4 mM). 3-Phosphoglycerate activites the enzyme (A_0.5 at about 0.3 mM) without causing a significant change in its K_m for fructose-2,6-P₂. The activities of both of these enzymes in relationship to the metabolic role of D-fructose 2,6-bisphosphate in the germinating seed is discussed.     

Promotion and inhibition of vesicle fusion by polylysine

Polylysine induced rapid aggregation of large unilamellar vesicles composed of phosphatidylcholine-cardiolipin (1:1 molar ratio) but not their fusion. Application of the terbium-dipicolinic acid fusion assay showed that addition of polylysine at nanomolar concentrations enabled a significant lowering of the Ca2+ threshold concentration for vesicle fusion from 9 to 1 mM. Analysis of the kinetics of fusion with a mass-action kinetic model showed that polylysine enhanced significantly the rate of aggregation but affected only slightly the rate of fusion per se. Maximal enhancement of overall fusion rates occurred at a charge ratio (polylysine/cardiolipin) of about 0.5. At larger polylysine concentrations, e.g., at charge ratios greater than 3, polylysine inhibited vesicle fusion.     

Focus: Zoonotic Disease: Zoonotic Ocular Onchocercosis by Onchocerca lupi

The parasitic filarioid Onchocerca lupi causes ocular disease characterized by conjunctivitis and nodular lesions. This nematode was first described in 1967 in a wolf from Georgia, and since then cases of infection from dogs and cats with ocular onchocercosis and sporadically from humans also with subcutaneous and cervical lesions caused by O. lupi have been reported from the Middle East, Europe, and North America. Due to its zoonotic potential, this parasitic infection has gained attention in the past 20 years. Phylogenetic studies have highlighted the recent divergence of O. lupi from other Onchocerca spp. and the importance of domestication in the evolutionary history of this worm. Moreover, the finding of an O. lupi genotype associated with subclinical and mild infection in the Iberian Peninsula, raises important questions about the pathogenicity of this presently enigmatic parasite.     

Pathology of Anopheles stephensi after infection with Plasmodium berghei berghei. II. Changes in amino acid contents.

Infection with Plasmodium berghei results in the disease of a relatively high percentage of mosquitoes depending on the experimental conditions. The damage caused by the parasites may be so severe that the host dies. It can also become manifest for instance in a change in the amino acid content of the mosquito homogenate. The amino acid content of mosquitoes fed on a glucose solution, normal mouse blood, or the blood of infected mice was analysed qualitatively and quantitatively over a period of 14 days. The amino acids lysine, phenylalanine, proline, threonine, and tyrosine are always found in higher concentrations in infected mosquitoes. The content of leucine (and/or isoleucine) increased from the 6th day and glutamic acid from the 9th day compared to the controls. Lower concentrations were found for alanine, aspartic acid, glycine, and serine as compared to uninfected mosquitoes. Further investigations on this subject might help to find the causes for the susceptibility or resistance of individual mosquitoes to plasmodia.     

Effect of Antiadherents on the Physical and Drug Release Properties of Acrylic Polymeric Films

Antiadherents are used to decrease tackiness of a polymer coating during both processing and subsequent storage. Despite being a common excipient in coating formulae, antiadherents may affect mechanical properties of the coating film as well as drug release from film-coated tablets, but how could addition of antiadherents affect these properties and to what extent and is there a relation between the physical characteristics of the tablet coat and the drug release mechanisms? The aim of this study was to evaluate physical characteristics of films containing different amounts of the antiadherents talc, glyceryl monostearate, and PlasACRYL^TM T20. Eudragit RL30D and Eudragit RS30D as sustained release polymers and Eudragit FS30D as a delayed release material were used. Polymer films were characterized by tensile testing, differential scanning calorimetry (DSC), microscopic examination, and water content as calculated from loss on drying. The effect of antiadherents on in vitro drug release for the model acetylsalicylic acid tablets coated with Eudragit FS30D was also determined. Increasing talc concentration was found to decrease the ability of the polymer films to resist mechanical stress. In contrast, glyceryl monostearate (GMS) and PlasACRYL produced more elastic films. Talc at concentrations higher than 25% caused negative effects, which make 25% concentration recommended to be used with acrylic polymers. All antiadherents delayed the drug release at all coating levels; hence, different tailoring of drug release may be achieved by adjusting antiadherent concentration with coating level.     

Direct observation of intraparticle equilibration and the rate-limiting step in adsorption of proteins in chromatographic adsorbents with confocal laser scanning microscopy

The adsorption of different proteins in a single biospecific and hydrophobic adsorbent particle for preparative protein chromatography has been observed directly by confocal laser scanning microscopy as a function of time at a constant bulk concentration c(b). The bulk concentration was in the non-linear part of the adsorption isotherm. At all times the concentration of free protein at the particle surface was almost equal to the bulk content indicating that external mass transfer resistance is not rate limiting for the adsorption under these conditions. Inside the particles a distinct maximum in adsorbed and free protein concentration that moved inside to a distance of approximately 0.2 R (R particle radius) from the particle surface, was observed. This is due to a decreasing solid-phase density and adsorptive capacity in the particle between 0.8 R and R indicating that the fraction of macropores (or void space) is larger in the outer than in the inner part of the adsorbent particles. By increasing the bulk concentration by a factor of 10 the equilibration time was reduced by about the same magnitude. This is in agreement with the concentration dependence of the effective pore diffusion coefficient D(p,eff)=D(p)/[epsilon(p)[1+nK/(K +c)(2)]] derived from the mass conservation relations describing the adsorption process. The time dependence protein adsorption up to approximately 90% of the equilibration value q* could be described by a bilinear free driving force model. The rapid equilibration in the outer part of the particle with a half-life time of approximately 100 s in the studied systems accounted for 0.3-0.4 q*. The slower equilibration with a up to ten times longer half-life time, was the adsorption in the inner part of the particle that outside 0.5 R accounts for 0.5-0.6 q*. These data were compared with literature data for batch adsorption of proteins in biospecific, hydrophobic and ion-exchange adsorbents. They could also be described by a bilinear free driving force model, with about the same quantitative results as obtained for similar conditions in the single particle experiments. The static adsorption parameters, maximum binding site concentration n, and dissociation constant for the protein binding to a binding site K, were determined from Scatchard plots. For the same protein-adsorbent system the plots changed from linear to non-linear with increasing n. This change occurred when the average distance between adjacent binding sites become of the same order of magnitude as the size of the binding site or adsorbed protein. This causes a shielding of free binding sites increasing with n and the concentration of adsorbed protein, yielding a concentration dependence in K. These results show that for a high throughput and rapid adsorption in preparative chromatography, the adsorption step should be carried out in the non-linear part of the adsorption isotherm with concentrations up to c(b) where q*/c(b)>/=10 to obtain high protein recoveries. To avoid tailing due to the flow of adsorbed proteins in the inner part of the particles further into the particles at the start of the desorption, and to speed up desorption rates, protein adsorption in the particle within 0.5 R from the particle center should be avoided. This requires the further development of suitable pellicular particles for preparative protein chromatography that meet this requirement.     

Identification of a novel type IV pilus gene cluster required for gastrointestinal colonization of Citrobacter rodentium

Citrobacter rodentium is used as an in vivo model system for clinically significant enteric pathogens such as enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). These pathogens all colonize the lumen side of the host gastrointestinal tract via attaching and effacing (A/E) lesion formation. In order to identify genes required for the colonization of A/E-forming pathogens, a library of signature-tagged transposon mutants of C. rodentium was constructed and screened in mice. Of the 576 mutants tested, 14 were attenuated in their ability to colonize the descending colon. Of these, eight mapped to the locus of enterocyte effacement (LEE), which is required for the formation of A/E lesions, underlying the importance of this mechanism for pathogenesis. Another mutant, P5H2, was found to have a transposon insertion in an open reading frame that has strong similarity to type IV pilus nucleotide-binding proteins. The region flanking the transposon insertion was sequenced, identifying a cluster of 12 genes that encode the first described pilus of C. rodentium (named colonization factor Citrobacter, CFC). The proteins encoded by cfc genes have identity to proteins of the type IV COF pilus of enterotoxigenic E. coli (ETEC), the toxin co-regulated pilus of Vibrio cholerae and the bundle-forming pilus of EPEC. A non-polar mutation in cfcI, complementation of this strain with wild-type cfcI and complementation of strain P5H2 with wild-type cfcH confirmed that these genes are required for colonization of the gastrointestinal tract by C. rodentium. Thus, CFC provides a convenient model to study type IV pilus-mediated pathogen-host interactions under physiological conditions in the natural colonic environment.     

Accumulation of plant galactolipid affects cell morphology of Escherichia coli

Monogalactosyldiacylglycerol (MGDG) is a major constituent of thylakoid membrane in chloroplasts. Therefore, it is considered to have an important role in the maintenance of the complicated structure of the thylakoid membrane. We have succeeded in cloning the enzyme for MGDG synthesis and overexpressed it in Escherichia coli. In this study we analyzed the morphology of the E. coli harboring the gene. The fatty acid composition of its membrane lipids did not differ between the wild type and transformant, except for the appearance of MGDG. However, transformant cells appeared to be elongated. DAPI staining revealed the entire intracellular region of filamentous cells to be stained; therefore, the elongation of the cells is probably due to a defect in cell division. Atomic force microscopy revealed that the transformant had a smooth but scratched surface. It was concluded that the excessive accumulation of a non-bilayer lipid, MGDG, interfered with the translocation of proteins across the plasma membrane, including those for cell division.     

Therapy of multiple myeloma

Multiple myeloma (MM) is a haematological malignancy characterised by the clonal expansion of malignant plasma cells within the bone marrow. It accounts for 10% of all haematological malignant diseases and 1% of all malignancies. The median age of patients at the time of the diagnosis is 70 years. The characteristic clinical features of MM are bone marrow failure, susceptibility to infections, bone pain, pathological bone fractures, hypercalcaemia, and renal failure. Though MM is currently incurable, the important progress in chemotherapy has resulted in an improvement in survival from a median of 7 months in the 1950-ies to about 3 years today. Advances in the diagnosis and in supportive treatment of infections, hypercalcaemia, and renal failure also contributed to the prolongation of survival. For decades, the gold standard of treatment had been oral melphalan alone or in combination with prednisolone. Combination chemotherapy has not improved overall survival (OS), but these regimens have led to the prolongation of event-free survival (EFS) and also to a better quality of life. High-dose chemotherapy with haemopoietic stem cell rescue resulted in a great improvement in EFS as well as OS. For those very few who have an HLA-compatible donor and are under 55, allogeneic bone marrow transplantation offers the best hope of survival but comes at a greatly increased risk of toxicity. There are conflicting data in the literature concerning the role of interferon-alpha; it seems to be able to prolong the duration of the plateau phase. Current treatment is moving towards an approach using sequential therapy. This involves induction therapy proceeding to high-dose chemotherapy with some form of stem-cell rescue. Bisphosphonates reduce hypercalcaemia, bone pain and can inhibit bone destruction. They also possess a direct antitumor activity. The better understanding of the pathomechanism of the disease gives the opportunity of the application of new therapeutic modalities such as antagonising the effect of interleukin-6 (IL-6), or idiotypic vaccination.