A twist code determines the onset of osteoblast differentiation

Runx2 is necessary and sufficient for osteoblast differentiation, yet its expression precedes the appearance of osteoblasts by 4 days. Here we show that Twist proteins transiently inhibit Runx2 function during skeletogenesis. Twist-1 and -2 are expressed in Runx2-expressing cells throughout the skeleton early during development, and osteoblast-specific gene expression occurs only after their expression decreases. Double heterozygotes for Twist-1 and Runx2 deletion have none of the skull abnormalities observed in Runx2(+/-) mice, a Twist-2 null background rescues the clavicle phenotype of Runx2(+/-) mice, and Twist-1 or -2 deficiency leads to premature osteoblast differentiation. Furthermore, Twist-1 overexpression inhibits osteoblast differentiation without affecting Runx2 expression. Twist proteins' antiosteogenic function is mediated by a novel domain, the Twist box, which interacts with the Runx2 DNA binding domain to inhibit its function. In vivo mutagenesis confirms the antiosteogenic function of the Twist box. Thus, relief of inhibition by Twist proteins is a mandatory event precluding osteoblast differentiation.     

Effects of ENU dosage on mouse strains

The germline supermutagen, N-ethyl-N-nitrosourea (ENU), has a variety of effects on mice. ENU is a toxin and carcinogen as well as a mutagen, and strains differ in their susceptibility to its effects. Therefore, it is necessary to determine an appropriate mutagenic, non-toxic dose of ENU for strains that are to be used in experiments. In order to provide some guidance, we have compiled data from a number of laboratories that have exposed male mice from inbred and non-inbred strains or their F₁ hybrids to ENU. The results show that most F₁ hybrid animals tolerate ENU well, but that inbred strains of mice vary in their longevity and in their ability to recover fertility after treatment with ENU. Received: 11 February 2000 / Accepted: 11 February 2000     

Mechanisms of lung endothelial barrier disruption induced by cigarette smoke: role of oxidative stress and ceramides

The epithelial and endothelial cells lining the alveolus form a barrier essential for the preservation of the lung respiratory function, which is, however, vulnerable to excessive oxidative, inflammatory, and apoptotic insults. Whereas profound breaches in this barrier function cause pulmonary edema, more subtle changes may contribute to inflammation. The mechanisms by which cigarette smoke (CS) exposure induce lung inflammation are not fully understood, but an early alteration in the epithelial barrier function has been documented. We sought to investigate the occurrence and mechanisms by which soluble components of mainstream CS disrupt the lung endothelial cell barrier function. Using cultured primary rat microvascular cell monolayers, we report that CS induces endothelial cell barrier disruption in a dose- and time-dependent manner of similar magnitude to that of the epithelial cell barrier. CS exposure triggered a mechanism of neutral sphingomyelinase-mediated ceramide upregulation and p38 MAPK and JNK activation that were oxidative stress dependent and that, along with Rho kinase activation, mediated the endothelial barrier dysfunction. The morphological changes in endothelial cell monolayers induced by CS included actin cytoskeletal rearrangement, junctional protein zonula occludens-1 loss, and intercellular gap formation, which were abolished by the glutathione modulator N-acetylcysteine and ameliorated by neutral sphingomyelinase inhibition. The direct application of ceramide recapitulated the effects of CS, by disrupting both endothelial and epithelial cells barrier, by a mechanism that was redox and apoptosis independent and required Rho kinase activation. Furthermore, ceramide induced dose-dependent alterations of alveolar microcirculatory barrier in vivo, measured by two-photon excitation microscopy in the intact rat. In conclusion, soluble components of CS have direct endothelial barrier-disruptive effects that could be ameliorated by glutathione modulators or by inhibitors of neutral sphingomyelinase, p38 MAPK, JNK, and Rho kinase. Amelioration of endothelial permeability may alleviate lung and systemic vascular dysfunction associated with smoking-related chronic obstructive lung diseases.     

Drosophila Ten-m and filamin affect motor neuron growth cone guidance

The Drosophila Ten-m (also called Tenascin-major, or odd Oz (odz)) gene has been associated with a pair-rule phenotype. We identified and characterized new alleles of Drosophila Ten-m to establish that this gene is not responsible for segmentation defects but rather causes defects in motor neuron axon routing. In Ten-m mutants the inter-segmental nerve (ISN) often crosses segment boundaries and fasciculates with the ISN in the adjacent segment. Ten-m is expressed in the central nervous system and epidermal stripes during the stages when the growth cones of the neurons that form the ISN navigate to their targets. Over-expression of Ten-m in epidermal cells also leads to ISN misrouting. We also found that Filamin, an actin binding protein, physically interacts with the Ten-m protein. Mutations in cheerio, which encodes Filamin, cause defects in motor neuron axon routing like those of Ten-m. During embryonic development, the expression of Filamin and Ten-m partially overlap in ectodermal cells. These results suggest that Ten-m and Filamin in epidermal cells might together influence growth cone progression.     

mGluR5 Ablation in Cortical Glutamatergic Neurons Increases Novelty-Induced Locomotion

The group I metabotropic glutamate receptor 5 (mGluR5) has been implicated in the pathology of various neurological disorders including schizophrenia, ADHD, and autism. mGluR5-dependent synaptic plasticity has been described at a variety of neural connections and its signaling has been implicated in several behaviors. These behaviors include locomotor reactivity to novel environment, sensorimotor gating, anxiety, and cognition. mGluR5 is expressed in glutamatergic neurons, inhibitory neurons, and glia in various brain regions. In this study, we show that deleting mGluR5 expression only in principal cortical neurons leads to defective cannabinoid receptor 1 (CB1R) dependent synaptic plasticity in the prefrontal cortex. These cortical glutamatergic mGluR5 knockout mice exhibit increased novelty-induced locomotion, and their locomotion can be further enhanced by treatment with the psychostimulant methylphenidate. Despite a modest reduction in repetitive behaviors, cortical glutamatergic mGluR5 knockout mice are normal in sensorimotor gating, anxiety, motor balance/learning and fear conditioning behaviors. These results show that mGluR5 signaling in cortical glutamatergic neurons is required for precisely modulating locomotor reactivity to a novel environment but not for sensorimotor gating, anxiety, motor coordination, several forms of learning or social interactions.     

Muscle activity and time to task failure differ with load compliance and target force for elbow flexor muscles

The primary purpose of this study was to determine the influence of load compliance on time to failure during sustained isometric contractions performed with the elbow flexor muscles at four submaximal target forces. Subjects pulled against a rigid restraint during the force task and maintained a constant elbow angle, while supporting an equivalent inertial load during the position task. Each task was sustained for as long as possible. Twenty-one healthy adults (23 ± 6 yr; 11 men) participated in the study. The maximal voluntary contraction (MVC) force was similar (P = 0.95) before the subjects performed the force and position tasks at each of the four target forces: 20, 30, 45, and 60% of MVC force. The time to task failure was longer for the force tasks (576 ± 80 and 325 ± 70 s) than for the position tasks (299 ± 77 and 168 ± 35 s) at target forces of 20 and 30% (P < 0.001), but was similar for the force tasks (178 ± 35 and 86 ± 14 s) and the position tasks (132 ± 29 and 87 ± 14 s) at target forces of 45 and 60% (P > 0.19). The briefer times to failure for the position task at the lower forces were accompanied by greater rates of increase in elbow flexor muscle activity, mean arterial pressure, heart rate, and rating of perceived exertion. There was no difference in the estimates of external mechanical work at any target force. The dominant mechanisms limiting time to failure of sustained isometric contractions with the elbow flexor muscles appear to change at target forces between 30 and 45% MVC, with load compliance being a significant factor at lower forces only.     

Process control in cell culture technology using dielectric spectroscopy

In the biopharmaceutical industry, mammalian and insect cells as well as plant cell cultures are gaining worldwide importance to produce biopharmaceuticals and as products themselves, for example in stem cell therapy. These highly sophisticated cell-based production processes need to be monitored and controlled to guarantee product quality and to satisfy GMP requirements. With the process analytical technology (PAT) initiative, requirements regarding process monitoring and control have changed and real-time in-line monitoring tools are now recommended. Dielectric spectroscopy (DS) can serve as a tool to satisfy some PAT requirements. DS has been used in the medical field for quite some time and it may allow real-time process monitoring of biological cell culture parameters. DS has the potential to enable process optimization, automation, cost reduction, and a more consistent product quality. Dielectric spectroscopy is reviewed here as a tool to monitor biochemical processes. Commercially available dielectric sensing systems are discussed. The potential of this technology is demonstrated through examples of current and potential future applications in research and industry for mammalian and insect cell culture.