To step or not to step? How biochemistry and mechanics influence processivity in Kinesin and Eg5

Conventional kinesin and Eg5 are essential nanoscale motor proteins. Single-molecule and presteady-state kinetic experiments indicate that both motors use similar strategies to generate movement along microtubules, despite having distinctly different in vivo functions. Single molecules of kinesin, a long-distance cargo transporter, are highly processive, binding the microtubule and taking 100 or more sequential steps at velocities of up to 700 nm/s before dissociating, whereas Eg5, a motor active in mitotic spindle assembly, is also processive, but takes fewer steps at a slower rate. By dissecting the structural, biochemical and mechanical features of these proteins, we hope to learn how kinesin and Eg5 are optimized for their specific biological tasks, while gaining insight into how biochemical energy is converted into mechanical work.     

Monkey locomotion during gait transitions: How do interlimb time intervals,step sequences,and kinematics change?

Cine film documenting unrestrained locomotion of vervet monkeys (Cercopithecus aethiops) ranging in age from 6 to greater than 48 months was analyzed to provide information on gait transitions from walking to loping. Changes in the duration of time between reciprocal footfalls were measured to determine how alternate limb movements, which occurred during walking, were converted to synchronous limb coordination characteristic of loping. Footfall pattern changes were also determined, and walk-lope transition speeds were plotted on logarithmic coordinates, as a function of body mass. Conversion from alternate to synchronous limb movement during vervet walk-lope transitions was effected by systematic decreases in the duration of time between successive footfalls. These decreases primarily affected contralateral limb pairs, RH-LH and RF-LF. Synchronous contralateral limb movement was considered to be mechanically advantageous because, when coupled with increased ranges of back motion, it provided a mechanism for increasing hindlimb step length. Intraspecific scaling of walk-lope transition speed in vervets provided support for McMahon's (1975) elastic similarity principle.     

Autophagy and self-preservation: a step ahead from cell plasticity?

Silencing the SPINK-related gene Kazal1 in hydra gland cells induces an excessive autophagy of both gland and digestive cells, leading to animal death. Moreover, during regeneration, autophagosomes are immediately detected in regenerating tips, where Kazal1 expression is lowered. When Kazal1 is completely silenced, hydra no longer survive the amputation stress (Chera S, de Rosa R, Miljkovic-Licina M, Dobretz K, Ghila L, Kaloulis K, Galliot B. Silencing of the hydra serine protease inhibitor Kazal1 gene mimics the human Spink1 pancreatic phenotype. J Cell Sci 2006; 119:846-57). These results highlight the essential digestive and cytoprotective functions played by Kazal1 in hydra. In mammals, autophagy of exocrine pancreatic cells is also induced upon SPINK1/Spink3 inactivation, whereas Spink3 is activated in injured pancreatic cells. Hence SPINKs, by preventing an excessive autophagy, appear to act as key players of the stress-induced self-preservation program. In hydra, this program is a prerequisite to the early cellular transition, whereby digestive cells of the regenerating tips transform into a head-organizer center. Enhancing the self-preservation program in injured tissues might therefore be the condition for unmasking their potential cell and/or developmental plasticity.     

One step synthesis of 6-oxo-cholestan-3β,5α-diol

Cholesterol metabolism has been recently linked to cancer, highlighting the importance of the characterization of new metabolic pathways in the sterol series. One of these pathways is centered on cholesterol-5,6-epoxides (5,6-ECs). 5,6-ECs can either generate dendrogenin A, a tumor suppressor present in healthy mammalian tissues, or the carcinogenic cholestane-3β,5α,6β-triol (CT) and its putative metabolite 6-oxo-cholestan-3β,5α-diol (OCDO) in tumor cells. We are currently investigating the identification of the enzyme involved in OCDO biosynthesis, which would be highly facilitated by the use of commercially unavailable [¹⁴C]-cholestane-3β,5α,6β-triol and [¹⁴C]-6-oxo-cholestan-3β,5α-diol. In the present study we report the one-step synthesis of [¹⁴C]-cholestane-3β,5α,6β-triol and [¹⁴C]-6-oxo-cholestan-3β,5α-diol by oxidation of [¹⁴C]-cholesterol with iodide metaperiodate (HIO₄).     

A-type lamin complexes and regenerative potential: a step towards understanding laminopathic diseases?

The lamins are nuclear intermediate filament-type proteins forming the nuclear lamina meshwork at the inner nuclear membrane as well as complexes in the nucleoplasm. The recent discoveries that mutated A-type lamins and lamin-binding nuclear membrane proteins can be linked to numerous rare human diseases (laminopathies) affecting a multitude of tissues has changed the cell biologist’s view of lamins as mere structural nuclear scaffold proteins. It is still unclear how mutations in these ubiquitously expressed proteins give rise to tissue-restricted pathological phenotypes. Potential disease models include mutation-caused defects in lamin structure and stability, the deregulation of gene expression, and impaired cell cycle control. This review brings together various previously proposed ideas and suggests a novel, more general, disease model based on an impairment of adult stem cell function and thus compromised tissue regeneration in laminopathic diseases.     

Purification of β-N-acetyl-d-glucosaminidase fromAspergillus niger using thermostabilization and heat as the initial step

A β-N-acetyl-d-glucosaminidase (EC 3.2.1.30) produced byAspergillus niger 419, was completely inactivated after heating 15 min at 65°C in 100 mM sodium phosphate buffer pH 7. The presence of 10% of polypropyleneglycol 1025 induced the thermal stability of the enzyme, the activity remaining unchanged after heating 60 min at 65°C. When this thermal treatment was used as the initial step of purification, the protein content of the crude extract was reduced by 98% without loss of the total initial enzymatic activity of the sample and a purification factor of 61. As the second and third step of purification DEAE-Sephacel, and Sephadex-G150 column chromatography were used, respectively. The final purification factor was 230 with a yield of 76%.     

Prolonged environmental stress via a two step process selects mutants of Escherichia,Salmonella and Pseudomonas that grow at 54°C

A prolonged incubation of Escherichia, Salmonella or Pseudomonas at 48°C with nalidixic acid selected mutants (T48) able to grow at 48°C. A prolonged incubation at 54°C of the T48 mutants selected mutants (T54) able to grow at 54°C. These mutants were susceptible to the same bacteriophages as the original mesophilic strains. Auxotrophic phenotypes of Escherichia coli and Salmonella typhimurium mesophilic parents were demonstrated by these mutants if they were cultivated on minimal agar with cellobiose at 48°C or 54°C or on a minimal agar with glucose at 37°C. The T48 alleles mapped in the gyrA region of E. coli or S. typhimurium chromosome. In S. typhimurium the T54 alleles, which permit growth at 54°C, were shown by cotransductional analysis to be linked to gyrA.     

Modern pollen and land-use relationships in the Taihang mountains, Hebei province, northern China—a first step towards quantitative reconstruction of human-induced land cover changes

Studies of the modern relationship between pollen, vegetation and land-use are essential to infer past human impact on vegetation from pollen records. Nevertheless, such investigations are relatively few in China. We present here a study of pollen assemblages from sediment samples collected from irrigation pools in the Tuoliang and Qipanshan catchments in northern China. Pollen and spores from natural vegetation such as Artemisia, Chenopodiaceae, Pinus and Selaginella sinensis dominate the pollen assemblages, while pollen types which could be from crops such as cereals, Brassicaceae, Fabaceae, Solanaceae, Apiaceae and Cucurbitaceae are common but not abundant. Pollen percentages of Artemisia and Chenopodiaceae become less with decreasing altitude, while Pinus and S. sinensis percentages increase, indicating that saccate Pinus pollen and S. sinensis spores are transported further than non-saccate Artemisia and Chenopodiaceae pollen, and differential sorting of pollen is occurring during transport in river water. Proportions of pollen from farmland and crops increase with decreasing altitude, showing that pollen percentages of crops might be a good indicator of the extent of farmland. A linear correlation analysis between pollen percentages and vegetation proportions shows that pollen percentages of crops are positively correlated with proportions of farmland, while correlation between pollen percentages of trees, shrubs and herbs and proportions of woodland, scrubland and grassland respectively is poor. This study indicates that the relationship between pollen percentages and vegetation proportions can be explained by the differences of pollen productivity, dispersal and deposition, and might be the basis for a modelling approach to infer past vegetation cover in northern China.     

Brassinosteroids, microtubules and cell elongation in Arabidopsis thaliana. I. Molecular, cellular and physiological characterization of the Arabidopsis bul1 mutant, defective in the Δ7-sterol-C5-desaturation step leading to brassinosteroid biosynthesis

Although cell elongation is a basic function of plant morphogenesis, many of the molecular events involved in this process are still unknown. In this work an extremely dwarf mutant, originally named bul, was used to study one of the main processes of plant development, cell elongation. Genetic analyses revealed that the BUL locus was linked to the nga172 marker on chromosome 3. Recently, after mapping the new dwf7 mutation of Arabidopsis, which is allelic to ste1, it was reported that dwf7 is also linked to the same marker. Sterol analyses of the bul1-1 mutant indicated that bul1-1 is defective in the Δ⁷-sterol-C5-desaturation step leading to brassinosteroid biosynthesis. Considering these findings, we designated our bul mutant as bul1-1/dwf7-3/ste1-4. The bul1-1 mutant was characterized by a very dwarf phenotype, with delayed development and reduced fertility. The mutant leaves had a dark-green colour, which was probably due to continuous stomatal closure. The bul1-1 mutant showed a partially de-etiolated phenotype in the dark. Cellular characterization and rescue experiments with brassinosteroids demonstrated the involvement of the BUL1-1 protein in brassinosteroid-dependent plant growth processes. Received: 28 April 2000 / Accepted: 6 October 2000     

Molecular carcinogenesis of hepatocellular carcinoma and intrahepatic cholangiocarcinoma: one step closer to personalized medicine?

Background

It remains unclear whether retroviruses can encode and express an intragenomic microRNA (miRNA). Some have suggested that processing by the Drosha and Dicer enzymes might preclude the viability of a replicating retroviral RNA genome that contains a cis-embedded miRNA. To date, while many studies have shown that lentiviral vectors containing miRNAs can transduce mammalian cells and express the inserted miRNA efficiently, no study has examined the impact on the replication of a lentivirus such as HIV-1 after the deliberate intragenomic insertion of a bona fide miRNA.

Results

We have constructed several HIV-1 molecular clones, each containing a discrete cellular miRNA positioned in Nef. These retroviral genomes express the inserted miRNA and are generally replication competent in T-cells. The inserted intragenomic miRNA was observed to elicit two different consequences for HIV-1 replication. First, the expression of miRNAs with predicted target sequences in the HIV-1 genome was found to reduce viral replication. Second, in one case, where an inserted miRNA was unusually well-processed by Drosha, this processing event inhibited viral replication.

Conclusion

This is the first study to examine in detail the replication competence of HIV-1 genomes that express cis-embedded miRNAs. The results indicate that a replication competent retroviral genome is not precluded from encoding and expressing a viral miRNA.     

How many and when? Optimising targeted gene flow for a step change in the environment

Targeted gene flow is an emerging conservation strategy that involves introducing individuals with particular traits to places where these traits are of benefit. One obvious application is to adapt a recipient population to a known threat, but questions remain as to how best to achieve this. Here, we vary timing and size of the introduction to maximise our objective – survival of the recipient population's genome. We explore a generic population model as well as a specific example – the northern quoll, an Australian marsupial predator threatened by the toxic cane toad. We reveal a trade‐off between preserving the recipient genome and reducing population extinction risk, but key management levers can often optimise this so that nearly 100% of the recipient population's genome is preserved. Any action was better than none but the size of the benefit was sensitive to outbreeding depression, recombination rate, and the timing and size of the introduction.     

Molecular carcinogenesis of hepatocellular carcinoma and intrahepatic cholangiocarcinoma: one step closer to personalized medicine?

Hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC) are the two major forms of primary liver cancers (PLC), accounting for approximately 90% and 5% respectively. The incidence of each is increasing rapidly in the western world, however our knowledge of the underlying mechanisms remains limited and the outcome, dismal. The etiologies of each vary geographically; nevertheless, chronic inflammation has been identified in more than 80% of the cases and appears to be a key mediator in altering the liver microenvironment, increasing the risk of carcinogenesis. However, since not all HCC and especially ICC cases have a recognized risk factor, there are currently two proposed models for liver carcinogenesis. The clonal evolution model demonstrates a multi-step process of tumor development from precancerous lesions to metastatic carcinoma, arising from the accumulation of genetic and epigenetic changes in a cell in the setting of chronic inflammation. While the majority of cases do occur as a consequence of chronic inflammation, most individuals with chronic infection do not develop PLC, suggesting the involvement of individual genetic and environmental factors. Further, since hepatocytes and cholangiocytes both have regenerative potential and arise from the same bi-potential progenitor cell, the more recently proposed cancer stem cell model is gaining its due attention. The integration of these models and the constant improvement in molecular profiling platforms is enabling a broader understanding of the mechanisms underlying these two devastating malignancies, perhaps moving us closer to a new world of molecularly-informed personalized medicine.     

A giant step towards artificial life?

Step by step, the components of an artificial form of cellular life are being assembled by researchers. Lipid vesicles the size of small bacteria can be prepared and under certain conditions are able to grow and divide, then grow again. Polymerase enzymes encapsulated in the vesicles can synthesize RNA from externally added substrates. Most recently, the entire translation apparatus, including ribosomes, has been captured in vesicles. Substantial amounts of proteins were produced, including green fluorescent protein used as a marker for protein synthesis. Can we now assemble a living cell? Not quite yet because no one has produced a polymerase that can be reproduced along with growth of the other molecular components required by life. But we are closer than ever before.