Fluorescent amino acids as versatile building blocks for chemical biology

Fluorophores have transformed the way we study biological systems, enabling non-invasive studies in cells and intact organisms, which increase our understanding of complex processes at the molecular level. Fluorescent amino acids have become an essential chemical tool because they can be used to construct fluorescent macromolecules, such as peptides and proteins, without disrupting their native biomolecular properties. Fluorescent and fluorogenic amino acids with unique photophysical properties have been designed for tracking protein–protein interactions in situ or imaging nanoscopic events in real time with high spatial resolution. In this Review, we discuss advances in the design and synthesis of fluorescent amino acids and how they have contributed to the field of chemical biology in the past 10 years. Important areas of research that we review include novel methodologies to synthesize building blocks with tunable spectral properties, their integration into peptide and protein scaffolds using site-specific genetic encoding and bioorthogonal approaches, and their application to design novel artificial proteins, as well as to investigate biological processes in cells by means of optical imaging. [Figure not available: see fulltext.]

The control of ovulation in mothers of dizygotic twins

Dizygotic twinning is familial, suggesting that there may be an inherited abnormality of the control of ovulation that predisposes to double ovulation and, therefore, dizygotic twins. The present study examines 17 mothers of dizygotic twins (MODZT) and 8 control mothers of singletons by daily blood sampling throughout an entire menstrual cycle. Blood samples were assayed for LH, FSH, estradiol, progesterone, and inhibin. The process of follicular development was followed by transvaginal ultrasound. The pituitary LH response to iv GnRH was also assessed. Three of the 16 MODZT double ovulated during the study compared to none of the 8 control mothers (P < 0.05). The number of small follicles (< 6 mm) declined significantly in control women at midcycle, but not in MODZT. There was no significant difference in serum FSH, LH, estradiol, or inhibin levels between the 2 groups at any stage of the menstrual cycle. During the follicular phase, serum progesterone levels were significantly higher in MODZT. The response to GnRH stimulation was not different between MODZT and controls. In conclusion, this study demonstrates an increased tendency to double ovulate in MODZT that may be due to a reduced rate of atresia in advanced follicles. Furthermore, the elevated progesterone levels in MODZT during the follicular phase suggest altered intrafollicular steroidogenesis that is independent of gonadotropins

CADM1 controls actin cytoskeleton assembly and regulates extracellular matrix adhesion in human mast cells

CADM1 is a major receptor for the adhesion of mast cells (MCs) to fibroblasts, human airway smooth muscle cells (HASMCs) and neurons. It also regulates E-cadherin and alpha6beta4 integrin in other cell types. Here we investigated a role for CADM1 in MC adhesion to both cells and extracellular matrix (ECM). Downregulation of CADM1 in the human MC line HMC-1 resulted not only in reduced adhesion to HASMCs, but also reduced adhesion to their ECM. Time-course studies in the presence of EDTA to inhibit integrins demonstrated that CADM1 provided fast initial adhesion to HASMCs and assisted with slower adhesion to ECM. CADM1 downregulation, but not antibody-dependent CADM1 inhibition, reduced MC adhesion to ECM, suggesting indirect regulation of ECM adhesion. To investigate potential mechanisms, phosphotyrosine signalling and polymerisation of actin filaments, essential for integrin-mediated adhesion, were examined. Modulation of CADM1 expression positively correlated with surface KIT levels and polymerisation of cortical F-actin in HMC-1 cells. It also influenced phosphotyrosine signalling and KIT tyrosine autophosphorylation. CADM1 accounted for 46% of surface KIT levels and 31% of F-actin in HMC-1 cells. CADM1 downregulation resulted in elongation of cortical actin filaments in both HMC-1 cells and human lung MCs and increased cell rigidity of HMC-1 cells. Collectively these data suggest that CADM1 is a key adhesion receptor, which regulates MC net adhesion, both directly through CADM1-dependent adhesion, and indirectly through the regulation of other adhesion receptors. The latter is likely to occur via docking of KIT and polymerisation of cortical F-actin. Here we propose a stepwise model of adhesion with CADM1 as a driving force for net MC adhesion