Rab GTPases containing a CAAX motif are processed post-geranylgeranylation by proteolysis and methylation

Post-translational modification by protein prenylation is required for membrane targeting and biological function of monomeric GTPases. Ras and Rho proteins possess a C-terminal CAAX motif (C is cysteine, A is usually an aliphatic residue, and X is any amino acid), in which the cysteine is prenylated, followed by proteolytic cleavage of the AAX peptide and carboxyl methylation by the Rce1 CAAX protease and Icmt methyltransferase, respectively. Rab GTPases usually undergo double geranylgeranylation within CC or CXC motifs. However, very little is known about processing and membrane targeting of Rabs that naturally contain a CAAX motif. We show here that a variety of Rab-CAAX proteins undergo carboxyl methylation, both in vitro and in vivo, with one exception. Rab38(CAKS) is not methylated in vivo, presumably because of the inhibitory action of the lysine residue within the AAX motif for cleavage by Rce1. Unlike farnesylated Ras proteins, we observed no targeting defects of overexpressed Rab-CAAX proteins in cells deficient in Rce1 or Icmt, as reported for geranylgeranylated Rho proteins. However, endogenous geranylgeranylated non-methylated Rab-CAAX and Rab-CXC proteins were significantly redistributed to the cytosol at steady-state levels and redistribution correlates with higher affinity of RabGDI for non-methylated Rabs in Icmt-deficient cells. Our data suggest a role for methylation in Rab function by regulating the cycle of Rab membrane recruitment and retrieval. Our findings also imply that those Rabs that undergo post-prenylation processing follow an indirect targeting pathway requiring initial endoplasmic reticulum membrane association prior to specific organelle targeting.     

Cold-stress-altered phosphorylation of EF-Tu in the cyanobacterium Anabaena sp. strain PCC 7120

Growth of prokaryotes at reduced temperature results in the formation of a cold-adapted ribosome through association with de novo synthesized polypeptides. In vitro and in vivo phosphorylation studies combined with affinity purification and mass spectrometry identified that the phosphorylation status of translation elongation factor EF-Tu was altered in response to cold stress in the photosynthetic, Gram-negative cyanobacterium Anabaena sp. strain PCC 7120. In response to a temperature downshift from 30 to 20 degrees C, EF-Tu was rapidly and transiently hyperphosphorylated during the acclimation phase followed by a reduction in phosphorylation below background levels in response to prolonged exposure. EF-Tu was identified as a phosphothreonine protein. Unexpectedly, ribosomal protein S2 was also observed to be a phosphoprotein continuously phosphorylated during cold stress. The phosphorylation status of EF-Tu has previously been associated with translational regulation in other systems, with a reduction in translation elongation occurring in response to phosphorylation. These results provide evidence for a novel mechanism by which translation is initially downregulated in response to cold stress in Anabaena.     

Drying Using Supercritical Fluid Technology as a Potential Method for Preparation of Chitosan Aerogel Microparticles

Supercritical fluid technology offers several advantages in preparation of microparticles. These include uniformity in particle size, morphology, and drug distribution without degradation of the product. One of the recent advantages is preparation of porous aerogel carrier with proper aerodynamic properties. In this study, we aimed to prepare chitosan aerogel microparticles using supercritical fluid (SCF) technology and compare that with microparticles produced by freeze drying (FD). Loading the prepared carriers with a model drug (salbutamol) was also performed. Comparisons of the particle properties and physicochemical characterizations were undertaken by evaluating particle size, density, specific surface area, and porosity. In vitro drug release studies were also investigated. The effect of many variables, such as molecular weight of chitosan oligomers, concentrations of chitosan, and concentrations of tripolyphosphate on the release, were also investigated. Chitosan aerogels were efficiently produced by SCF technology with an average particle size of 10 μm with a tapped density values around 0.12 g/mL, specific surface area (73–103) m²/g, and porosity (0.20–0.29) cc/g. Whereas, microparticles produced by FD method were characterized as cryogels with larger particle size (64 microns) with clear cracking at the surface. Sustained release profile was achieved for all prepared microparticles of salbutamol produced by the aforementioned methods as compared with pure drug. The results also demonstrates that chitosan molecular weight, polymer concentration, and tripolyphosphate concentration affected the release profile of salbutamol from the prepared microparticles. In conclusion, SCF technology was able to produce chitosan aerogel microparticles loaded with salbutamol that could be suitable for pulmonary drug delivery system.KEY WORDS: aerodynamic, aerogels, chitosan, salbutamol, supercritical fluid technology     

The effect of gamma irradiation on potato microtuber production in vitro

The effects of low doses of gamma irradiation and potato (Solanum tuberosum L.) cultivar on the production of microtubers in vitro were investigated. Nodal segments from virus free explants of three potato cultivars (cv.) were placed on tuberization inducing medium and irradiated with 4 doses of gamma radiation (2.5, 5, 10, 15 Gy). Cv. Diamant produced the highest number of microtubers followed by Draga and Spunta. Irradiation of the explants with 2.5 Gy of gamma radiation led to a significant increase in the number of microtubers (38% increase over the control). Average weight of microtubers was not significantly influenced by low doses of gamma irradiation. Draga microtubers were the largest followed by Diamant and Spunta. Microtubers resembled mature tubers in shape (Spunta was oval and Draga and Diamant were spherical). Size of microtubers was crucial for sprouting in vivo. It is suggested that only microtubers larger than 5 mm in diameter (250 mg) be used to produce minitubers in vivo. Since 2.5 Gy is a low irradiation dose, it can be used to enhance tuberization in vitro without fear of genetic changes in the used cultivars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Synthesis of an iron(II)-braced proline-II tripod protein

This paper describes the engineering of braced tripod proteins for use as molecular frameworks. Specifically, a 30-residue tripod-shaped protein with three proline-II helical legs braced by an iron(II)tris(bipyridine) complex was modularly designed, chemically synthesized, and biophysically characterized. Three copies of a 10-residue leg peptide were covalently linked through sulfide bonds to an N-terminal apex (1,3,5-tris(methylene)benzene) and by amide bonds to the brace (FeII(Mbc)3: Mbc is 4-methyl-2,2-bipyridine-4-carbonyl). The leg peptide (H-Cys-Pro5-Pra(Mbc)-Pro3-NH2: Pra is cis-4-amino-l-proline) was assembled by the solid-phase method using Boc-Pra(Mbc)-OH, which was synthesized in 75% overall yield by coupling Mbc-OH to the 4-amino group of Boc-Pra-OCH3 and saponifying the methyl ester group.The iron(II)-braced tripod was assembled by S-alkylation of three copies of the leg peptide with 1,3,5-tris(bromomethyl)benzene followed by ligation of Fe2+ to the resulting unbraced tripod. The CD spectrum of the iron(II)-braced tripod showed a positive MLCT band at 570 nm and a negative –* band at 312 nm, so its FeII(Mbc)3 brace was predominantly in the configuration. In a mostly acetonitrile solution at 25 °C, the leg peptide and the unbraced tripod isomerized from the proline-II helical form into the proline-I helical form but the iron(II)-braced tripod remained in the proline-II helical form.     

GlbN (cyanoglobin) is a peripheral membrane protein that is restricted to certain Nostoc spp.

The glbN gene of Nostoc commune UTEX 584 is juxtaposed to nifU and nifH, and it encodes a 12-kDa monomeric hemoglobin that binds oxygen with high affinity. In N. commune UTEX 584, maximum accumulation of GlbN occurred in both the heterocysts and vegetative cells of nitrogen-fixing cultures when the rate of oxygen evolution was repressed to less than 25 micromol of O2 mg of chlorophyll a(-1) h(-1). Accumulation of GlbN coincided with maximum synthesis of NifH and ferredoxin NADP+ oxidoreductase (PetH or FNR). A total of 41 strains of cyanobacteria, including 40 nitrogen fixers and representing 16 genera within all five sections of the cyanobacteria were screened for the presence of glbN or GlbN. glbN was present in five Nostoc strains in a single copy. Genomic DNAs from 11 other Nostoc and Anabaena strains, including Anabaena sp. strain PCC 7120, provided no hybridization signals with a glbN probe. A constitutively expressed, 18-kDa protein which cross-reacted strongly with GlbN antibodies was detected in four Anabaena and Nostoc strains and in Trichodesmium thiebautii. The nifU-nifH intergenic region of Nostoc sp. strain MUN 8820 was sequenced (1,229 bp) and was approximately 95% identical to the equivalent region in N. commune UTEX 584. Each strand of the DNA from the nifU-nifH intergenic regions of both strains has the potential to fold into secondary structures in which more than 50% of the bases are internally paired. Mobility shift assays confirmed that NtcA (BifA) bound a site in the nifU-glbN intergenic region of N. commune UTEX 584 approximately 100 bases upstream from the translation initiation site of glbN. This site showed extensive sequence similarity with the promoter region of glnA from Synechococcus sp. strain PCC 7942. In vivo, GlbN had a specific and prominent subcellular location around the periphery of the cytosolic face of the cell membrane, and the protein was found solely in the soluble fraction of cell extracts. Our hypothesis is that GlbN scavenges oxygen for and is a component of a membrane-associated microaerobically induced terminal cytochrome oxidase.     

Fast and sensitive silver staining of DNA in polyacrylamide gels.

The photochemically derived silver stain of nucleic acids in polyacrylamide gels originally described by Merril et al. (1981, Science 211, 1437-1438) was modified to reduce unspecific background staining and increase sensitivity (down to 1 pg/mm2 band cross-section). Detection limits for double-stranded DNA fragments from HaeIII endonuclease digests of phage phi X174 were maintained despite eliminating oxidation pretreatment of fixed gels and reducing silver nitrate concentration. Preexposure to formaldehyde during silver impregnation enhanced sensitivity and the inclusion of the silver-complexing agent sodium thiosulphate in the image developer decreased background staining. Higher formaldehyde concentration during image development resulted in darker bands with good contrast. The procedure almost halves the number of steps, solutions and experimental time required and can be used for the staining of DNA fragments in polyacrylamide gels bound to a polyester backing film by controlling temperature during image development. We have applied this improved staining procedure for the routine analysis of complex DNA profiles generated by DNA amplification fingerprinting (DAF).     

Assessment of the bacterial contamination of hand air dryer in washrooms

The present study was carried out, using standard techniques, to identify and count the bacterial contamination of hand air dryers, used in washrooms. Bacteria were isolated from the air flow, outlet nozzle of warm air dryers in fifteen air dryers used in these washrooms. Bacteria were found to be relatively numerous in the air flows. Bacterially contaminated air was found to be emitted whenever a warm air dryer was running, even when not being used for hand drying. Our investigation shows that Staphylococcus haemolyticus, Micrococcus luteus, Pseudomonas alcaligenes, Bacillus cereus and Brevundimonad diminuta/vesicularis were emitted from all of the dryers sampled, with 95% showing evidence of the presence of the potential pathogen S. haemolyticus. It is concluded that hot air dryers can deposit pathogenic bacteria onto the hands and body of users. Bacteria are distributed into the general environment whenever dryers are running and could be inhaled by users and none-users alike. The results provide an evidence base for the development and enhancement of hygienic hand drying practices.     

Bismuth-inhibitory effects on bacteria and stimulation of fungal growth in vitro

Bismuth salicylate was found to inhibit the growth of a range of bacteria and yeast, “Candida albicans”. In general the growth of bacteria did not result in increase in bismuth solubilisation, in contrast, bismuth solubilisation increased following the growth of C. albicans. A significant increase in the biomass (dry weight) of Aspergillus niger and Aspergillus oryzae occurred in vitro when these fungi were grown in the presence of bismuth salicylate. Biomass increase occurred over a range of bismuth compound additions, which in the case of A. oryzae was associated with increase in the solubilisation of the insoluble bismuth compounds.