The flagella of ‘Candidatus Liberibacter asiaticus’ and its movement in planta

Citrus huanglongbing (HLB) is the most devastating citrus disease worldwide. ‘Candidatus Liberibacter asiaticus’ (Las) is the most prevalent HLB causal agent that is yet to be cultured. Here, we analysed the flagellar genes of Las and Rhizobiaceae and observed two characteristics unique to the flagellar proteins of Las: (i) a shorter primary structure of the rod capping protein FlgJ than other Rhizobiaceae bacteria and (ii) Las contains only one flagellin-encoding gene flaA (CLIBASIA_02090), whereas other Rhizobiaceae species carry at least three flagellin-encoding genes. Only flgJ_Atu but not flgJ_Las restored the swimming motility of Agrobacterium tumefaciens flgJ mutant. Pull-down assays demonstrated that FlgJ_Las interacts with FlgB but not with FliE. Ectopic expression of flaA_Las in A. tumefaciens mutants restored the swimming motility of ∆flaA mutant and ∆flaAD mutant, but not that of the null mutant ∆flaABCD. No flagellum was observed for Las in citrus and dodder. The expression of flagellar genes was higher in psyllids than in planta. In addition, western blotting using flagellin-specific antibody indicates that Las expresses flagellin protein in psyllids, but not in planta. The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control.     

Cytochemical demonstration of catalasepositive particles (peroxisomes?) in fibroblasts

Summary The connective tissue of the mucosa of the respiratory tract, of the gastric mucosa and of the mucosa of the tongue was investigated in mice. The tissue was fixed in glutaraldehyde and incubated in an alkaline DAB-medium to demonstrate the peroxidatic activity of catalase. In fibroblasts and fibrocytes, as well as in lymphoid cells, membrane bounded particles from 0.10 to 0.25 m in diameter were found, whose matrices were intensely stained by the histochemical reaction. The reaction is inhibited by the addition of 2×10^–2 M 3-amino-1,2,4-triazole. In connective tissue cells of specimens, which were not reacted to demonstrate catalase activity, these organelles show a granular matrix of moderate electron density. They lack a crystalline core. The possibility that these catalase-positive particles (CPs) represent peroxisomes is discussed.This investigation was kindly supported by a grant of Hochschuljubiläumsstiftung der Stadt Wien.     

“Intracisternal microtubules” in neurons of dog ganglia

Summary The changes which occur in the ultrastructure of the mesothoracic retractor unguis muscle of the cockroach Periplaneta americana as a result of disuse are described. Breakdown of myofibrils, sarcoplasmic reticulum, and mitochondria are all marked, this degeneration only being apparent 9 weeks after the operation.     

Effect of sildenafil citrate (Viagra?) on trace element concentration in serum and brain of rats

As a vasodilator with good hemodynamic effects, sildenafil has been successfully used in the treatment of patients with pulmonary hypertension and cardiovascular diseases. By selectively inhibiting phosphodiestrase type 5 (PDE-5) and thus effectively reducing the breakdown of c GMP, sildenafil administration can markedly improve the erectile dysfunction. Sildenafil also elevates localized cerebral blood flow in rat brain. The objective of the present study was to investigate the effect of sildenafil on the level of trace elements (Zinc (Zn), copper (Cu), iron (Fe), selenium (Se), cobalt (Co), and chromium (Cr)) in blood and brain of rats. Sixteen male albino rats weighing 180-200 g were divided into two groups (8 rats/group). Sildenafil (Viagra, Pfizer Inc.) was dissolved in saline and administered at a dose of 10mg/kg i.p. (0.5 ml volume) to rats in the treated group every 72 h for 12 injections. Rats in the control group were administered the same volume of saline as in treated group. All rats were sacrificed 24h after the last injection. Blood samples were collected and serum was separated and stored at -20°C. Brains were dissected and stored frozen until analysis. Trace elements concentrations were determined by flame emission atomic absorption spectrophotometer. Results showed that sildenafil injection significantly (P<0.05) increased serum and brain Se and Cu concentrations. Moreover, sildenafil increased the Cr concentration in the brain tissue. It was concluded that sildenafil citrate administration increased serum Se and Cu as well as, increased brain Se, Cu, and Cr concentrations in rats.     

What generates flux of tubulin in kinetochore microtubules?

Summary. We discuss models for production of tubulin flux in kinetochore microtubules. Current models concentrate solely on microtubules and their associated motors and enzymes. For example, in some models the driving force for flux is enzymes at the poles and the kinetochores; in others the driving force is motor molecules that are associated with a stationary spindle matrix. We present a different viewpoint, that microtubules are propelled poleward by forces arising from the spindle matrix, that the forces on the microtubules “activate” polymerising and depolymerising enzymes at kinetochores and poles, that matrix forces utilise actin, myosin, and microtubule motors, and that the matrix itself may not necessarily be static. Correspondence: A. Forer, Biology Department, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.     

Evidence for three “classes” of microtubules in the interpolar space of the mitotic micronucleus of a ciliate and the participation of the nuclear envelope in conferring stability to microtubules

Exposure of Nyctotherus ovalis to low temperatures or vinblastine caused similar reactions of classes of microtubules (mt) present in the mitotic micronucleus of this ciliate towards both treatments. However, differences of sensitivity between certain classes of mt at individual mitotic stages exist. Unlike the kinetochore mt (kmt) of most other eukaryotic cells, kmt in Nyctotherus completely disassemble after incubation at 6–8 ° C (60 min) and most disappear after prolonged exposure to vinblastine (10^–5 M, 16 h). The depolymerization of kmt causes the collapse of the spindle and a dislocation of chromosomes at metaphase, yet the reduced number of kmt after vinblastine-treatment still allows an alignment of composite complexes at the spindle equator. The data suggest that three individual sets of mt exist in the interpolar spindle region during ana- and telophase: 1) interpolar mt (int mt), which are assembled during anaphase, are cold- and vinblastine sensitive; 2) manchette mt (ma mt), which are first observed underneath the nuclear envelope during mid-anaphase, are cold-stable and insensitive to vinblastine treatment (10^–5 M); after prolonged treatment (16 h) they form spiral structures; 3) stembody mt (st mt), comprising the interpolar region of the nucleus during telophase, are cold- and vinblastine insensitive. Paracrystalline structures resembling a stembody are formed in telophase-like division stages after prolonged vinblastine exposure (16 h, 10^–5 M). Since kmt and int mt possess the same sensitivity under depolymerizing conditions, they probably have a similar composition. Thus the idea that the int mt in this organism arise by elongation of kmt is supported. However, st mt apparently do not originate from an extension of preexistent int mt, but appear to represent a new set of stable mt. This is emphasized not only by their greater stability compared to the int mt but also by the distribution of cold-stable mt in late anaphase micronuclei. The ma mt may be an intermediary step in formation of st mt since their stability resembles that of the st mt. A comparison of the substructure of vinblastine-induced paracrystals in Nyctotherus with those observed in in vitro systems with known composition suggests that a turnover of MAPs may be responsible for the different stability of mt and thus could specify and regulate mt sensitivity and function. Another organelle, possibly involved in conferring stability to mt, is the nuclear membrane. The assumption that the nuclear envelope possesses an intrinsic property to nucleate mt and thus aid in the alignment of mt is supported.     

PEG-ing down (and preventing?) the cause of pegloticase failure

Pegloticase is a powerful but underutilized weapon in the rheumatologist’s armamentarium. The drug’s immunogenicity leads to neutralizing antibody formation and rapid loss of efficacy in roughly one-half of all patients, which remains an impediment to broader use. New data, however, suggest that drug survival might improve with concomitant immunosuppressive agent (s), which merits further study. Efficacy appears to be unchanged when pegloticase is infused at 3-week (rather than 2-week) intervals. Stretching the time between infusions may also improve patient adherence and allow for earlier identification of transient responders.In the previous issue of Arthritis Research and Therapy, Hershfield and colleagues published a study putting forth a number of novel and potentially important findings regarding pegloticase, a powerful but underutilized weapon in the small but growing anti-hyperuricemic arsenal [1].The study examined the efficacy of pegloticase in a cohort of 30 patients with severe gout (93% tophaceous) utilizing an every 3-week infusion regimen, rather than the every 2-week schedule employed in previously published phase 3 trials. Despite the longer interval between infusions in the current study, the effectiveness of pegloticase is no worse (17/30 patients are persistent responders), and the pharmacokinetics of the drug suggest this should come as no surprise. The authors correctly note that a 3-week interval would be significantly more convenient for patients, and notably that such a regimen would also prove less costly to payors. Hershfield and colleagues’ dosing schedule would also help to identify transient responders earlier in the course of treatment – only four of 12 transient responders had uric acid >6 mg/dl 2 weeks after the first infusion (three of whom had previously been exposed to pegloticase in earlier phase 1 and 2 studies), whereas 11 of 12 transient responders had uric acid >6 mg/dl at 3 weeks (vs. only one of 17 persistent responders). For the reasons just elaborated upon, the paper demonstrates that dosing every 3 weeks may not just be as good as the current protocol, but may in some ways be superior.Hershfield and colleagues also upend the assumption that neutralizing antibodies to pegloticase are formed against uricase itself. Their paper clearly demonstrates that neutralizing antibodies develop in response to the polyethylene glycol (PEG) moiety of the drug, a finding that rebuffs a recent commentary which suggested antibodies to PEG are not pathogenic [2]. A septe and larger study (169 patients exposed to pegloticase) published in the previous issue Arthritis Research and Therapy by Lipsky and colleagues reaches the same conclusion regarding anti-pegloticase antibodies: anti-PEG antibodies are responsible for loss of efficacy rather than antibodies to the uricase enzyme itself, the latter of which rarely occur (positive more than once in only 11 subjects) and occur much later during the course of treatment, long after neutralizing anti-pegloticase antibodies have developed [3]. This larger study also demonstrates that an anti-pegloticase antibody titer >1:2,430 generally predicts loss of efficacy to the drug.Finally, and not least of all, Hershfield and colleagues’ smaller study included post-transplant patients (who were excluded from the phase 3 studies), a population particularly susceptible to developing gout. Of seven post-transplant subjects in the study, six proved to be persistent responders (86%) [1]. Although this is an admittedly small number of patients upon which to base any conclusion, it does raise the intriguing question of whether immunosuppression might lead to less of a mounted antibody response against pegloticase, and thus to more favorable outcomes. This is no small point; patients who are placed on pegloticase generally have severe, long-standing, and refractory gout, and should be given every chance to optimize their response to a potentially transformative therapy.While this signal is worth pursuing, some questions are immediately raised: how immunosuppressed must patients be to prevent neutralizing anti-PEG antibody formation, and with what should this be accomplished? Of the small subcohort in this trial, all patients were on cyclosporine or mycophenolate mofetil, and five of seven patients were on a combination of immunosuppressive agents. In choosing an immunosuppressant for the express purpose of preventing neutralizing antibodies, the risk of these drugs would very probably outweigh any proposed benefit (cyclosporine in particular might be the least desirable immunosuppressant for a patient with severe gout, because it both increases serum uric acid levels and decreases the glomerular filtration rate).If a trial was designed to investigate this line of query, a reasonable immunosuppressive agent of choice might be methotrexate. This drug has been shown to effectively prevent neutralizing antibodies from forming against monoclonal antibodies to anti-tumor necrosis factor [4,5]. Methotrexate’s inhibitory effect may not extend to preventing antibody formation against PEG, although methotrexate has also been shown to inhibit antibody formation against the polysaccharide 23-valent pneumococcal vaccine [6]. Methotrexate might also yield the unintended benefit of acting as an anti-inflammatory agent to suppress gouty attacks. However, because patients with severe gout have multiple comorbidities that place them at higher risk for medication side effects, methotrexate should not be considered in the clinical setting in the absence of data to support its use [7]. Nevertheless, methotrexate therapy is an avenue of inquiry that is clinically relevant and needs exploration to increase the likelihood that patients who begin this powerful drug can remain on it.     

Phylogeny of the Alismatales (Monocotyledons) and the relationship of Acorus (Acorales?)

A phylogenetic analysis of the early branching lineages of the monocotyledons is performed using data from two plastid genes (rbcL and matK), five mitochondrial genes (atp1, ccmB, cob, mttB and nad5) and morphology. The complete matrix includes 93 terminals representing Acorus, the 14 families currently recognized within Alismatales, and numerous lineages of monocotyledons and other angiosperms. Total evidence analysis results in an almost completely resolved strict consensus tree, but all data partitions, genomic as well as morphological, are incongruent. The effects of RNA editing and potentially processed paralogous sequences are explored and discussed. Despite a decrease in incongruence length differences after exclusion of edited sites, the major data partitions remain significantly incongruent. The 14 families of Alismatales are all found to be monophyletic, but Acorus is found to be included in Alismatales rather than being the sister group to all other monocotyledons. The placement is strongly supported by the mitochondrial data, atp1 in particular, but it cannot be explained as an artifact caused by patterns of editing or by sampling of processed paralogues.     

Polar organizers and girdling bands of microtubules are associated with γ-tubulin and act in establishment of meiotic quadripolarity in the hepatic Aneura pinguis (Bryophyta)

Summary. Meiosis in Aneura pinguis is preceded by extensive cytoplasmic preparation for quadripartitioning of the diploid sporocyte into a tetrad of haploid spores. In early prophase the four future spore domains are defined by lobing of the cytoplasm and development of a quadripolar prophase spindle focused at polar organizers (POs) centered in the lobes. Cells entering the reproductive phase become isolated and, instead of hooplike cortical microtubules, have endoplasmic microtubule systems centered on POs. These archesporial cells proliferate by mitosis before entering meiosis. In prophase of each mitosis, POs containing a distinct concentration of γ-tubulin appear de novo at tips of nuclei and initiate the bipolar spindle. Cells entering meiosis become transformed into quadrilobed sporocytes with four POs, one in each lobe. This transition is a complex process encompassing assembly of two opposite POs which subsequently disperse into intersecting bands of microtubules that form around the central nucleus. The girdling bands define the future planes of cytokinesis and the cytoplasm protrudes through the restrictive bands becoming quadrilobed. Two large POs reappear in opposite cleavage furrows. Each divides and the resulting POs migrate into the tetrahedral lobes of cytoplasm. Cones of microtubules emanating from the four POs interact to form a quadripolar microtubule system (QMS) that surrounds the nucleus in meiotic prophase. The QMS is subsequently transformed into a functionally bipolar metaphase spindle by migration of poles in pairs to opposite cleavage furrows. These findings contribute to knowledge of microtubule organization and the role of microtubules in spatial regulation of cytokinesis in plants. Correspondence and reprints: Department of Biology, University of Louisiana-Lafayette, Lafayette, LA 70504-2451, U.S.A.     

Single-particle imaging reveals intraflagellar transport–independent transport and accumulation of EB1 in Chlamydomonas flagella

The microtubule (MT) plus-end tracking protein EB1 is present at the tips of cilia and flagella; end-binding protein 1 (EB1) remains at the tip during flagellar shortening and in the absence of intraflagellar transport (IFT), the predominant protein transport system in flagella. To investigate how EB1 accumulates at the flagellar tip, we used in vivo imaging of fluorescent protein–tagged EB1 (EB1-FP) in Chlamydomonas reinhardtii. After photobleaching, the EB1 signal at the flagellar tip recovered within minutes, indicating an exchange with unbleached EB1 entering the flagella from the cell body. EB1 moved independent of IFT trains, and EB1-FP recovery did not require the IFT pathway. Single-particle imaging showed that EB1-FP is highly mobile along the flagellar shaft and displays a markedly reduced mobility near the flagellar tip. Individual EB1-FP particles dwelled for several seconds near the flagellar tip, suggesting the presence of stable EB1 binding sites. In simulations, the two distinct phases of EB1 mobility are sufficient to explain its accumulation at the tip. We propose that proteins uniformly distributed throughout the cytoplasm like EB1 accumulate locally by diffusion and capture; IFT, in contrast, might be required to transport proteins against cellular concentration gradients into or out of cilia.     

Molecular characterization of heat shock proteins 90 (HSP83?) and 70 in tropical strains of Bombyx mori

Following the concept of whole organism, we have extracted total protein from the Bombyx mori for the identification and analysis of HSPs. Expression of 90 kDa HSP in first, second and third instars, 84 kDa in fourth instar and 90‐, 84‐, 62‐, 60‐, 52‐ and 33‐kDa HSPs in fifth instar larvae of tropical polyvoltine and bivoltine silkworm strains were obvious. Further, we have combined single and 2‐DE with MALDI‐TOF for analysis of BmHSPs. Ninety kilodalton band excised from 1‐DE gel was identified as HSP83 by MALDI‐TOF‐MS. The immunoblot analysis confirmed the expression of HSP90 in all the instars larvae of B. mori. Heat shock‐induced protein spots were excised from 2‐DE gels for MALDI‐TOF‐MS analysis. The Mascot search results are for HSP68, HSC70‐1 and HSP70Ba in Pure Mysore, and major HSP70Bbb, HSP68, HSC‐3 and HSP83 in NB₄D₂. Multiple sequence alignment explicit the variations in amino acid sequence between Pure Mysore and NB₄D₂. Notably, the PMF of spot 2 matched the coding sequence of B. mori and its gene annotation was determined on chromosome 9. With this novel approach, expression of BmHSP90 was confirmed in all the instars and uncovered isoforms of BmHSP70, which provided unequivocal insight to analyze and understand the biological significance in B. mori.     

EMG in rotation–flexion of the torso

The objective of this study was to determine the magnitude and phasic relationship of the torso muscles in rotation–flexion of varying degree of asymmetries of the trunk. Nineteen normal young subjects (7 males and 12 females) were stabilized on a posture stabilizing platform and instructed to assume a flexed and right rotated posture. A combination 20°, 40° and 60° of rotation and 20°, 40° and 60° of flexion resulted in nine postures. These postures were assumed in a random order. The subjects were asked to exert their maximal voluntary isometric contraction (MVC) in the plane of rotation of the posture assumed for a period of 5 s. The surface EMG from the external and internal obliques, rectus abdominis, latissimus dorsi and erector spinae at the 10th thoracic and 3rd lumbar vertebral levels was recorded. The abdominal muscles had the least response at 40° of flexion, the dorsal muscles had the highest magnitude.With increasing right rotation, the left external oblique continued to decrease its activity. The ANOVA revealed that rotation and muscles had a significant main effect on normalized peak EMG (p < 0.02) in both genders. There was a significant interaction between rotation and flexion in both genders (p < 0.02) and rotation and muscle in females. The erector spinae activity was highest at 40° flexion, due to greater mechanical disadvantage and having not reached the state of flexion–relaxation. The abdominal muscle activity declined with increasing asymmetry, due to the decreasing initial muscle length. The EMG activity was significantly affected by rotation than flexion (p < 0.02).     

Forensic cultures in historical perspective: Technologies of witness,testimony, judgment (and justice?)

This article explores the history of forensic science in terms of ideologies and institutions rather than developing technique. It presents an analytical framework for characterising forensic institutions and practices, past and present. That framework highlights the distinct issues of means of witness, accredited testimony, and the reaching of juridical decisions. The article applies the framework by comparing four forensic ‘formations,’ (or ‘cultures’) which have been prominent at various times and places in the western world from the early modern period onward: these are the central European heritage of the Caroline code, a eugenically-oriented forensic enterprise of late nineteenth-century America, the forensic perspective in nineteenth-century British India, and the representation of forensic certainty in contemporary American popular culture. The article concludes with a critique of what seems an increasingly common expectation: that forensic science evolves independently of legal institutions, and can ultimately displace them.     

Oxyphenbutazone (Tandearil?) in Rhinoplasty—A Clinical Evaluation of Effectiveness

To evaluate the effectiveness of Oxyphenbutazone as an anti-inflammatory agent, a double-blind study of Oxyphenbutazone and a placebo in a group of 42 patients who had nasal cosmetic operations involving osteotomy was carried out. The observations included direct objective measurement of the width of the palpebral fissure after operation, grading of the severity of postoperative edema and ecchymosis from photographs, and observations by the patients regarding the clearing of the postoperative discoloration. It appeared from the results of these observations that Oxyphenbutazone is not effective in preventing postoperative edema in such operations or in promoting more rapid resolution of postoperative edema. It did appear to enhance the clearing of postoperative periorbital ecchymosis.     

Posttranslational acetylation of α-tubulin constrains protofilament number in native microtubules

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Highlights? α-TAT mutants have short microtubules and variable protofilament number ? α-tubulin K40 acetylation promotes interprotofilament salt bridges ? α-tubulin K40 acetylation is a key constraint on protofilament number in vivo     

Of mice and (Viking?) men: phylogeography of British and Irish house mice

The west European subspecies of house mouse (Mus musculus domesticus) has gained much of its current widespread distribution through commensalism with humans. This means that the phylogeography of M. m. domesticus should reflect patterns of human movements. We studied restriction fragment length polymorphism (RFLP) and DNA sequence variations in mouse mitochondrial (mt) DNA throughout the British Isles (328 mice from 105 localities, including previously published data). There is a major mtDNA lineage revealed by both RFLP and sequence analyses, which is restricted to the northern and western peripheries of the British Isles, and also occurs in Norway. This distribution of the 'Orkney' lineage fits well with the sphere of influence of the Norwegian Vikings and was probably generated through inadvertent transport by them. To form viable populations, house mice would have required large human settlements such as the Norwegian Vikings founded. The other parts of the British Isles (essentially most of mainland Britain) are characterized by house mice with different mtDNA sequences, some of which are also found in Germany, and which probably reflect both Iron Age movements of people and mice and earlier development of large human settlements. MtDNA studies on house mice have the potential to reveal novel aspects of human history.     

Is the interaction of CNP with tubulin and microtubules required for process extension in oligodendrocytes?

The STOP protein (stable tubule-only polypeptide) is a calmodulin-regulated protein which associates with microtubules and induces cold stabilization. There are different isoforms of this protein that arise from alternative splicing of STOP mRNA. Neurons express two major variants N-STOP (125 kDa) and E-STOP (84 kDa). NIH 3T3 fibroblasts contain a major F-STOP isoform (42 kDa) and two minor STOP variants (48 and 89 kDa). Previously, we demonstrated the presence of N-STOP in the cytoskeleton associated with myelin isolated from animals injected with apotransferrin. Since this protein was only described as a neuronal protein we decided to further investigate the expression of this protein in oligodendrocyte cultures. The analysis of the STOP protein expression in oligodendrocyte shows that STOP protein is expressed in the soma and processes of oligodendrocyte precursors, as well as in immature and mature oligodendroglial cells. In addition, we found that MBP shows a high degree of colocalization with STOP protein. By Western blot analysis, it was found that these cells express a major STOP variant (89 kDa). When the cultures were exposed to cold temperature we found that STOP protein associates with microtubules and induces microtubule cold stabilization. Under these experimental conditions, we found that MBP associates with microtubules too, and maintains its colocalization with STOP protein. At present, we are doing new assays directed to further characterize STOP (89 kDa) protein and to elucidate how this protein participates in the formation of myelin by oligodendrocytes.