Combining Laser Microsurgery and Finite Element Modeling to Assess Cell-Level Epithelial Mechanics

Laser microsurgery and finite element modeling are used to determine the cell-level mechanics of the amnioserosa—a morphogenetically crucial epithelium on the dorsal surface of fruit fly embryos (Drosophila melanogaster). In the experiments, a tightly focused laser ablates a subcellular hole (1 μm in diameter) that passes clean through the epithelium. The surrounding cells recoil from the wound site with a large range of initial recoil velocities. These depend on the embryo's developmental stage and the subcellular wound site. The initial recoil (up to 0.1 s) is well reproduced by a base finite element model, which assumes a uniform effective viscosity inside the cells, a constant tension along each cell-cell boundary, and a large, potentially anisotropic, far-field stress—one that far exceeds the stress equivalent of the cell-edge tensions. After 0.1 s, the experimental recoils slow dramatically. This observation can be reproduced by adding viscoelastic rods along cell edges or as a fine prestressed mesh parallel to the apical and basal membranes of the cell. The mesh also reproduces a number of double-wounding experiments in which successive holes are drilled in a single cell.     

BMP signaling modulates hedgehog-induced secondary heart field proliferation

Sonic hedgehog signaling in the secondary heart field has a clear role in cardiac arterial pole development. In the absence of hedgehog signaling, proliferation is reduced in secondary heart field progenitors, and embryos predominantly develop pulmonary atresia. While it is expected that proliferation in the secondary heart field would be increased with elevated hedgehog signaling, this idea has never been tested. We hypothesized that up-regulating hedgehog signaling would increase secondary heart field proliferation, which would lead to arterial pole defects. In culture, secondary heart field explants proliferated up to 6-fold more in response to the hedgehog signaling agonist SAG, while myocardial differentiation and migration were unaffected. Treatment of chick embryos with SAG at HH14, just before the peak in secondary heart field proliferation, resulted unexpectedly in stenosis of both the aortic and pulmonary outlets. We examined proliferation in the secondary heart field and found that SAG-treated embryos exhibited a much milder increase in proliferation than was indicated by the in vitro experiments. To determine the source of other signaling factors that could modulate increased hedgehog signaling, we co-cultured secondary heart field explants with isolated pharyngeal endoderm or outflow tract and found that outflow tract co-cultures prevented SAG-induced proliferation. BMP2 is made and secreted by the outflow tract myocardium. To determine whether BMP signaling could prevent SAG-induced proliferation, we treated explants with SAG and BMP2 and found that BMP2 inhibited SAG-induced proliferation. In vivo, SAG-treated embryos showed up-regulated BMP2 expression and signaling. Together, these results indicate that BMP signaling from the outflow tract modulates hedgehog-induced proliferation in the secondary heart field.     

Cardiac arterial pole alignment is sensitive to FGF8 signaling in the pharynx

Morphogenesis of the cardiac arterial pole is dependent on addition of myocardium and smooth muscle from the secondary heart field and septation by cardiac neural crest cells. Cardiac neural crest ablation results in persistent truncus arteriosus and failure of addition of myocardium from the secondary heart field leading to malalignment of the arterial pole with the ventricles. Previously, we have shown that elevated FGF signaling after neural crest ablation causes depressed Ca2+ transients in the primary heart tube. We hypothesized that neural crest ablation results in elevated FGF8 signaling in the caudal pharynx that disrupts secondary heart field development. In this study, we show that FGF8 signaling is elevated in the caudal pharynx after cardiac neural crest ablation. In addition, treatment of cardiac neural crest-ablated embryos with FGF8b blocking antibody or an FGF receptor blocker rescues secondary heart field myocardial development in a time- and dose-dependent manner. Interestingly, reduction of FGF8 signaling in normal embryos disrupts myocardial secondary heart field development, resulting in arterial pole malalignment. These results indicate that the secondary heart field myocardium is particularly sensitive to FGF8 signaling for normal conotruncal development, and further, that cardiac neural crest cells modulate FGF8 signaling in the caudal pharynx.     

Human mitochondrial branched chain aminotransferase isozyme: structural role of the CXXC center in catalysis

Mammalian branched chain aminotransferases (BCATs) have a unique CXXC center. Kinetic and structural studies of three CXXC center mutants (C315A, C318A, and C315A/C318A) of human mitochondrial (hBCATm) isozyme and the oxidized hBCATm enzyme (hBCATm-Ox) have been used to elucidate the role of this center in hBCATm catalysis. X-ray crystallography revealed that the CXXC motif, through its network of hydrogen bonds, plays a crucial role in orienting the substrate optimally for catalysis. In all structures, there were changes in the structure of the beta-turn preceding the CXXC motif when compared with wild type protein. The N-terminal loop between residues 15 and 32 is flexible in the oxidized and mutant enzymes, the disorder greater in the oxidized protein. Disordering of the N-terminal loop disrupts the integrity of the side chain binding pocket, particularly for the branched chain side chain, less so for the dicarboxylate substrate side chain. The kinetic studies of the mutant and oxidized enzymes support the structural analysis. The kinetic results showed that the predominant effect of oxidation was on the second half-reaction rather than the first half-reaction. The oxidized enzyme was completely inactive, whereas the mutants showed limited activity. Model building of the second half-reaction substrate alpha-ketoisocaproate in the pyridoxamine 5'-phosphate-hBCATm structure suggests that disruption of the CXXC center results in altered substrate orientation and deprotonation of the amino group of pyridoxamine 5'-phosphate, which inhibits catalysis.     

An Investigation of the Locomotor Function of Therian Forearm Pronation Provides Renewed Support for an Arboreal,Chameleon-like Evolutionary Stage

Although investigations of forelimb characteristics are central to therian evolutionary studies, the functional origins of forearm pronation are neglected. However, recent research based on bipedal manipulations strongly suggests that proximal radioulnar joint mobility is highly conserved in tetrapods. This new information calls for a replication of previously published physical simulations of forearm bone movements, to investigate whether active therian pronation/supination evolved from the plesiomorphic mechanism via which locomotor-induced torsion is passively alleviated during forelimb retraction. Preliminary results using representative extant and extinct tetrapod forelimb elements are supportive, and also offer insight into why another overlooked forearm trait, osteological full pronation (mechanically aligned elbow and wrist/finger joints), evolved only in therians and chameleons. During forelimb retraction in tetrapods with unfused radii/ulnae, the radius unexpectedly remains fixed in place as a functional complex with the firmly planted manus/carpus, which the ulnar complex (ulna/humerus) displaces relative to. Therefore, the highly conserved functional morphology of the tetrapod forearm indicates that enhanced therian manual dexterity, which emphasizes isolated radial movements bipedally, was preceded by the locomotor evolution of ulnar supination relative to the radius quadrupedally. This counterintuitive information indicates that the traditional hypothesis, that therian pronation/supination evolved arboreally to amplify radial mobility, requires modification. The authors propose that proximal long-axis rotations of the therian ulnar complex co-evolved with osteological full pronation during a period of arboreal, chameleon-like locomotion, to continue allowing torsion at a reinforced proximal radioulnar joint. These adaptations were later or simultaneously co-opted for object manipulation using active radioulnar pronation/supination.     

A simple apparatus for ultrafiltration of small volumes: application to the measurement of free and albumin-bound tryptophan in plasma.

An ultrafiltration apparatus which is cheap, easy to make, and simple to operate has been developed (patent pending) and assessed for the measurement of nonprotein-bound tryptophan concentration in blood plasma. Large batches of 200-μl samples can be rapidly ultrafiltered at controlled pH. The apparatus is of potentially wide applicability to protein-binding studies and to the determination of free and bound concentrations of drugs and other substances in plasma.     

A technique for monitoring mammalian cell growth and inhibition in situ via Fourier transform infrared spectroscopy

A single culture of Chinese hamster ovary cells was grown on germanium attenuated total reflectance (ATR) crystals and continuously monitored in situ via ATR/Fourier transform infrared (FT-IR) spectroscopy for approximately 60 h. The cells were seeded into a specially designed flow cell which controlled physiological conditions, flow rate, and addition of growth medium or metabolic inhibitors. Infrared spectra were taken at 20-min intervals until a confluent monolayer was formed. Several strong bands are evident in the spectra which can be generally ascribed to molecular features of cellular components. Cell growth kinetics were measured as a function of infrared band intensity over time and exhibited the normal lag phase, logarithmic growth, and stationary phase on reaching confluence. Spectra of growing cells, normalized to the area under the spectral region 1800-1000 cm-1, were subtracted from reference spectra of confluent cells at 60 h. Difference spectra showed that the largest differences were observed between confluent cells and cells in early growth stages. Differences may reflect cell morphological changes, biochemical activity, and degree of ATR crystal exposure to the bulk medium. ATR/FT-IR spectroscopy of living Chinese hamster ovary cells was also used in a toxicological study to monitor the effects of hydroxyurea, an inhibitor of DNA synthesis. Delayed growth was observed in the cell growth curve of the hydroxyurea-treated cells during the course of treatment as compared to the control culture.