An important goal of nanotechnology is the application of individual molecule handling techniques to the discovery of potential new therapeutic agents. Of particular interest is the search for new inhibitors of metabolic routes exclusive of human pathogens, such as the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway essential for the viability of most human pathogenic bacteria and of the malaria parasite. Using atomic force microscopy single-molecule force spectroscopy (SMFS), we have probed at the single-molecule level the interaction of 1-deoxy-D-xylulose 5-phosphate synthase (DXS), which catalyzes the first step of the MEP pathway, with its two substrates, pyruvate and glyceraldehyde-3-phosphate. The data obtained in this pioneering SMFS analysis of a bisubstrate enzymatic reaction illustrate the substrate sequentiality in DXS activity and allow for the calculation of catalytic parameters with single-molecule resolution. The DXS inhibitor fluoropyruvate has been detected in our SMFS competition experiments at a concentration of 10 µM, improving by 2 orders of magnitude the sensitivity of conventional enzyme activity assays. The binding of DXS to pyruvate is a 2-step process with dissociation constants of koff = 6.1 x 10–4 ± 7.5 x 10–3 and 1.3 x 10–2 ± 1.0 x 10–2 s–1, and reaction lengths of xβ = 3.98 ± 0.33 and 0.52 ± 0.23 Å. These results constitute the first quantitative report on the use of nanotechnology for the biodiscovery of new antimalarial enzyme inhibitors and open the field for the identification of compounds represented only by a few dozens of molecules in the sensor chamber.—Sisquella, X., de Pourcq, K., Alguacil, J., Robles, J., Sanz, F., Anselmetti, D., Imperial, S., Fernàndez-Busquets, X. A single-molecule force spectroscopy nanosensor for the identification of new antibiotics and antimalarials.
Day: 11/01/2010
Unique and redundant functions of integrins in the epidermis
The skin forms a barrier against the environment and protects us from mechanical trauma, pathogens, radiation, dehydration, and dangerous temperature fluctuations. The epithelium of the skin, the epidermis, is in a continuous equilibrium of growth and differentiation and has the remarkable capacity to self-renew completely, which relies on reservoirs of stem cells. Epidermal homeostasis is further dependent on proper repair after injury, and on tight adhesion to the underlying basement membrane. Epidermal adhesion is mediated primarily by integrins, cell-surface receptors that connect the extracellular matrix to the cytoskeleton. In addition, numerous in vitro reports have implicated integrins, integrin-associated proteins, or downstream integrin effectors in the regulation of a plethora of cellular processes other than adhesion. Over the past decade, a wealth of information on the function of these proteins has been gathered both from (conditional) knockout mice and from human skin disorders, allowing for a reconstruction of integrin signaling pathways in vivo. Here, we address how epidermal integrins and integrin-associated proteins regulate keratinocyte adhesion, proliferation, and differentiation, as well as signal transduction, re-epithelialization during wound healing, hair growth, and stem cell maintenance. Furthermore, we discuss human pathologies associated with altered integrin functions in the epidermis.—Margadant, C., Charafeddine, R. A., Sonnenberg, A. Unique and redundant functions of integrins in the epidermis.
adhesion • hair • hemidesmosome • stem cells • wound healing
Lenalidomide in Nonmetastatic Biochemically Relapsed Prostate Cancer
To evaluate the safety and activity of 6 months of treatment with lenalidomide at 5 or 25 mg/d in nonmetastatic biochemically relapsed prostate cancer.
Experimental Design: Sixty men with non-castrate, nonmetastatic, biochemically relapsed prostate cancer were stratified by prostate-specific antigen (PSA) doubling time, surgery/radiation therapy, prior androgen deprivation therapy (ADT), and randomized to lenalidomide 5 mg (n = 26) or 25 mg/d (n = 34) for 3 weeks repeated monthly for 6 months or until dose-limiting toxicity or disease progression. Toxicity was evaluated monthly, and PSAs and X-rays/scans every 6 months. Study size was determined to detect a progression rate of 40% at 6 months in either arm with 85% power (compared with a rate of 80% in the population receiving no treatment). Changes in PSA slopes were calculated using the regression of the log PSA for each patient before and during the initial 6 months and compared by t test.
Results: Baseline variables were balanced between arms. Grade 3/4 toxicity rates were 12% (n = 3) with 5 mg and 29% (n = 10) with 25 mg (P = 0.1), most commonly neutropenia (five patients, all on 25 mg). Two patients per arm had thromboembolic events. The change in PSA slope was greater with 25 mg versus 5 mg [−0.172 (−0.24 to −0.11) versus −0.033 (−0.11 to 0.04); P = 0.005]. With a mean follow-up of 31.4 months (range 14-44), five patients on 25 mg and one patient on 5 mg remain on the study.
Conclusions: Lenalidomide has acceptable toxicity and is associated with long-term disease stabilization and PSA declines. Randomized studies evaluating conventional clinical disease end points in this patient population are planned.
ARYAN'S BLOG 