Disorganization of cortical microtubules stimulates tangential expansion and reduces the uniformity of cellulose microfibril alignment among cells in the root of Arabidopsis

To test the role of cortical microtubules in aligning cellulose microfibrils and controlling anisotropic expansion, we exposed Arabidopsis thaliana roots to moderate levels of the microtubule inhibitor, oryzalin. After 2 d of treatment, roots grow at approximately steady state. At that time, the spatial profiles of relative expansion rate in length and diameter were quantified, and roots were cryofixed, freeze-substituted, embedded in plastic, and sectioned. The angular distribution of microtubules as a function of distance from the tip was quantified from antitubulin immunofluorescence images. In alternate sections, the overall amount of alignment among microfibrils and their mean orientation as a function of position was quantified with polarized-light microscopy. The spatial profiles of relative expansion show that the drug affects relative elongation and tangential expansion rates independently. The microtubule distributions averaged to transverse in the growth zone for all treatments, but on oryzalin the distributions became broad, indicating poorly organized arrays. At a subcellular scale, cellulose microfibrils in oryzalin-treated roots were as well aligned as in controls; however, the mean alignment direction, while consistently transverse in the controls, was increasingly variable with oryzalin concentration, meaning that microfibril orientation in one location tended to differ from that of a neighboring location. This conclusion was confirmed by direct observations of microfibrils with field-emission scanning electron microscopy. Taken together, these results suggest that cortical microtubules ensure microfibrils are aligned consistently across the organ, thereby endowing the organ with a uniform mechanical structure.     

Genomic and morphological evidence converge to resolve the enigma of Strepsiptera

The phylogeny of insects, one of the most spectacular radiations of life on earth, has received considerable attention. However, the evolutionary roots of one intriguing group of insects, the twisted-wing parasites (Strepsiptera), remain unclear despite centuries of study and debate. Strepsiptera exhibit exceptional larval developmental features, consistent with a predicted step from direct (hemimetabolous) larval development to complete metamorphosis that could have set the stage for the spectacular radiation of metamorphic (holometabolous) insects. Here we report the sequencing of a Strepsiptera genome and show that the analysis of sequence-based genomic data (comprising more than 18 million nucleotides from nearly 4,500 genes obtained from a total of 13 insect genomes), along with genomic metacharacters, clarifies the phylogenetic origin of Strepsiptera and sheds light on the evolution of holometabolous insect development. Our results provide overwhelming support for Strepsiptera as the closest living relatives of beetles (Coleoptera). They demonstrate that the larval developmental features of Strepsiptera, reminiscent of those of hemimetabolous insects, are the result of convergence. Our analyses solve the long-standing enigma of the evolutionary roots of Strepsiptera and reveal that the holometabolous mode of insect development is more malleable than previously thought.     

Association of the Golgi UDP-galactose transporter with UDP-galactose:ceramide galactosyltransferase allows UDP-galactose import in the endoplasmic reticulum

UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose:ceramide galactosyltransferase. It is not known how UDP-galactose is transported from the cytosol into the endoplasmic reticulum. We transfected ceramide galactosyltransferase cDNA into CHOlec8 cells, which have a defective UGT and no endogenous ceramide galactosyltransferase. Cotransfection with the human UGT1 greatly stimulated synthesis of lactosylceramide in the Golgi and of galactosylceramide in the endoplasmic reticulum. UDP-galactose was directly imported into the endoplasmic reticulum because transfection with UGT significantly enhanced synthesis of galactosylceramide in endoplasmic reticulum membranes. Subcellular fractionation and double label immunofluorescence microscopy showed that a sizeable fraction of ectopically expressed UGT and ceramide galactosyltransferase resided in the endoplasmic reticulum of CHOlec8 cells. The same was observed when UGT was expressed in human intestinal cells that have an endogenous ceramide galactosyltransferase. In contrast, in CHOlec8 singly transfected with UGT 1, the transporter localized exclusively to the Golgi complex. UGT and ceramide galactosyltransferase were entirely detergent soluble and form a complex because they could be coimmunoprecipitated. We conclude that the ceramide galactosyltransferase ensures a supply of UDP-galactose in the endoplasmic reticulum lumen by retaining UGT in a molecular complex.     

Ticks of the Central African Republic

A review is presented of the ticks of the Central African Republic (C.A.R.), based on unpublished data and a literature review. We consider as established in the C.A.R. two species of Argasidae (one species of each of the genera Carios and Ornithodoros) and 49 species of Ixodidae: 5 Ixodes, 11 Amblyomma, 2 Dermacentor, 9 Haemaphysalis, 4 Hyalomma, and 18 Rhipicephalus species, 3 of which belong to the subgenus Boophilus. Two of these species, one Amblyomma and one Dermacentor, may by now have disappeared from the country together with their hosts, the black and white rhinoceroses. Moreover, four other species of Ixodidae have been recorded, but are not necessarily established in the country, and 6 species of Ixodidae have apparently been reported incorrectly; the data for the occurrence of three other species of Ixodidae are uncertain. Rhipicephalus species in the C.A.R. present the greatest difficulties in identification, on the one hand because of uncertainties in taxonomy, particularly in the capensis group, on the other hand because of individual variability, which means that individual specimens cannot always be reliably identified to species. Examining the dissected and cleared gonopore structure of females helps, but is very time-consuming where large numbers are involved. Some of the species of the genus Haemaphysalis are also difficult to identify with certainty. There are only a few species of Hyalomma in the country. We revised also some old records of ticks in C.A.R., which we consider as unreliable and should be disregarded because of subsequent changes in taxonomy and nomenclature, unless the material is still available for examination.     

Protein C-Terminal Labeling and Biotinylation Using Synthetic Peptide and Split-Intein

Background

Site-specific protein labeling or modification can facilitate the characterization of proteins with respect to their structure, folding, and interaction with other proteins. However, current methods of site-specific protein labeling are few and with limitations, therefore new methods are needed to satisfy the increasing need and sophistications of protein labeling.

Methodology

A method of protein C-terminal labeling was developed using a non-canonical split-intein, through an intein-catalyzed trans-splicing reaction between a protein and a small synthetic peptide carrying the desired labeling groups. As demonstrations of this method, three different proteins were efficiently labeled at their C-termini with two different labels (fluorescein and biotin) either in solution or on a solid surface, and a transferrin receptor protein was labeled on the membrane surface of live mammalian cells. Protein biotinylation and immobilization on a streptavidin-coated surface were also achieved in a cell lysate without prior purification of the target protein.

Conclusions

We have produced a method of site-specific labeling or modification at the C-termini of recombinant proteins. This method compares favorably with previous protein labeling methods and has several unique advantages. It is expected to have many potential applications in protein engineering and research, which include fluorescent labeling for monitoring protein folding, location, and trafficking in cells, and biotinylation for protein immobilization on streptavidin-coated surfaces including protein microchips. The types of chemical labeling may be limited only by the ability of chemical synthesis to produce the small C-intein peptide containing the desired chemical groups.     

Evolution of enzymatic activity in the tautomerase superfamily: mechanistic and structural studies of the 1,3-dichloropropene catabolic enzymes

The use of the soil fumigant Telone II, which contains a mixture of cis- and trans-1,3-dichloropropene, to control plant-parasitic nematodes is a common agricultural practice for maximizing yields of various crops. The effectiveness of Telone II is limited by the rapid turnover of the dichloropropenes in the soil due to the presence of bacterial catabolic pathways, which may be of recent origin. The characterization of three enzymes in these pathways, trans-3-chloroacrylic acid dehalogenase (CaaD), cis-3-chloroacrylic acid dehalogenase (cis-CaaD), and malonate semialdehyde decarboxylase (MSAD), has uncovered intriguing catalytic mechanisms as well as a fascinating evolutionary lineage for these proteins. Sequence comparisons and mutagenesis studies revealed that all three enzymes belong to the tautomerase superfamily. Tautomerase superfamily members with known structures are characterized by a β-α-β structural fold. Moreover, they have a conserved N-terminal proline, which plays an important catalytic role. Mechanistic, NMR, and pH rate studies of the two dehalogenases, coupled with a crystal structure of CaaD inactivated by 3-bromopropiolate, indicate that they use a general acid/base mechanism to catalyze the conversion of their respective isomer of 3-chloroacrylate to malonate semialdehyde. The reaction is initiated by the conjugate addition of water to the C-2, C-3 double bond and is followed by the loss of HCl. MSAD processes malonate semialdehyde to acetaldehyde, and is the first identified decarboxylase in the tautomerase superfamily. The catalytic mechanism is not well defined but the N-terminal proline plays a prominent role and may function as a general acid catalyst, similar to its role in CaaD and cis-CaaD. These are the first structural and mechanistic details for tautomerase superfamily members that catalyze either a hydration or a decarboxylation reaction, rather than a tautomerization reaction, in which Pro-1 serves as a general acid catalyst rather than as a general base catalyst. The available information on the 1,3-dichloropropene catabolic enzymes allows speculation on the possible evolutionary origins of their activities.     

Arsenic trioxide stimulates SUMO-2/3 modification leading to RNF4-dependent proteolytic targeting of PML

We have recently reported that poly-SUMO-2/3 conjugates are subject to a ubiquitin-dependent proteolytic control in human cells. Here we show that arsenic trioxide (ATO) increases SUMO-2/3 modification of promyelocytic leukemia (PML) leading to its subsequent ubiquitylation in vivo. The SUMO-binding ubiquitin ligase RNF4 mediates this modification and causes disruption of PML nuclear bodies upon treatment with ATO. Reconstitution of SUMO-dependent ubiquitylation of PML by RNF4 in vitro and in a yeast trans vivo system revealed a preference of RNF4 for chain forming SUMOs. Polysumoylation of PML in response to ATO thus leads to its recognition and ubiquitylation by RNF4.     

Prolonged blood glucose reduction in mrp-2 deficient rats (GY/TR(-)) by the glucose-6-phosphate translocase inhibitor S 3025

Chlorogenic acid derivatives are potent inhibitors of hepatic glucose production by inhibition of the glucose-6-phosphate translocase component of the hepatic glucose-6-phosphatase system. The pharmacological proof of concept was clearly demonstrated during i.v. infusion of potent derivatives (S 4048, S 3483) in rats. However, the blood glucose lowering effect of S 4048 after bolus i.v. injection lasted only 60-90 min. Plasma clearance of S 4048 was very high, and the parent compound was rapidly and efficiently excreted into the bile of Wistar and GY/TR(-) rats, indicating that mrp-2 was not involved in this hepatobiliary elimination process. About 72% of the total administered radioactivity appeared in the bile within 20 min after i.v. bolus injection of the radiolabeled analogue [(3)H]S 1743 in a Wistar rat. However, in GY/TR(-) rats the dicarboxylic analogue of S 4048, S 3025, was cleared from the plasma less rapidly than its parent compound and its biliary elimination was comparatively low. In contrast, S 3025 exhibited comparable pharmacokinetics and biliary elimination profile as S 4048 in Wistar rats, suggesting that biliary elimination of S 3025 is facilitated by mrp-2, functionally absent in GY/TR(-) rats. Targeting to mrp-2 resulted in a significantly prolonged reduction of blood glucose levels in GY/TR(-) rats after i.v. bolus administration of S 3025.     

Freeze-thaw tolerance and clues to the winter survival of a soil community

Although efforts have been made to sample microorganisms from polar regions and to investigate a few of the properties that facilitate survival at freezing or subzero temperatures, soil communities that overwinter in areas exposed to alternate freezing and thawing caused by Foehn or Chinook winds have been largely overlooked. We designed and constructed a cryocycler to automatically subject soil cultures to alternating freeze-thaw cycles. After 48 freeze-thaw cycles, control Escherichia coli and Pseudomonas chlororaphis isolates were no longer viable. Mixed cultures derived from soil samples collected from a Chinook zone showed that the population complexity and viability were reduced after 48 cycles. However, when bacteria that were still viable after the freeze-thaw treatments were used to obtain selected cultures, these cultures proved to be >1,000-fold more freeze-thaw tolerant than the original consortium. Single-colony isolates obtained from survivors after an additional 48 freeze-thaw cycles were putatively identified by 16S RNA gene fragment sequencing. Five different genera were recognized, and one of the cultures, Chryseobacterium sp. strain C14, inhibited ice recrystallization, a property characteristic of antifreeze proteins that prevents the growth of large, potentially damaging ice crystals at temperatures close to the melting temperature. This strain was also notable since cell-free medium derived from cultures of it appeared to enhance the multiple freeze-thaw survival of another isolate, Enterococcus sp. strain C8. The results of this study and the development of a cryocycler should allow further investigations into the biochemical and soil community adaptations to the rigors of a Chinook environment.     

The effect of immunomodulators on the immunogenicity of TNF-blocking therapeutic monoclonal antibodies: a review

Therapeutic monoclonal antibodies have revolutionized the treatment of various inflammatory diseases. Immunogenicity against these antibodies has been shown to be clinically important: it is associated with shorter response duration because of diminishing concentrations in the blood and with infusion reactions. Concomitant immunomodulators in the form of methotrexate or azathioprine reduced the immunogenicity of therapeutic antibodies in rheumatoid arthritis, Crohn disease, and juvenile idiopathic arthritis. The occurrence of adverse events does not increase when immunomodulators are added to therapeutic antibodies. The mechanism whereby methotrexate and azathioprine influence immunogenicity remains unclear. Evidence-based consensus on prescribing concomitant immunomodulators is needed.