Dissection and characterization of microtubule bundles in the mitotic spindle using femtosecond laser ablation.

The mitotic spindle is a highly organized and dynamic structure required for segregation of the genetic material into two daughter cells. Although most of the individual players involved in building the spindle have been characterized in vitro, a general understanding of how all of the spindle players act together in vivo is still missing. Hence, in recent years, experiments have focused on introducing mechanical perturbations of the spindle on a micron scale, thereby providing insight into its function and organization, as well as into forces acting in the spindle. Among different types of mechanical perturbations, optical ones are more flexible, less invasive, and more precise than other approaches. In this chapter, we describe a detailed protocol for cutting the microtubule bundles in human cells using a near-infrared femtosecond laser. This type of laser microsurgery provides the ability to precisely sever a single microtubule bundle while preserving spindle integrity and dynamics. Furthermore, we describe quantitative measurements obtained from the response of a severed microtubule bundle to laser ablation, which reveal the structure and function of individual parts of the spindle, such as the bridging fiber connecting sister k-fibers. Finally, the method described here can be easily combined with other quantitative techniques to address the complexity of the spindle.

Modeling the insulin-glucose feedback system: the significance of pulsatile insulin secretion.

A mathematical model of the insulin-glucose feedback regulation in man is used to examine the effects of an oscillatory supply of insulin compared to a constant supply at the same average rate. We show that interactions between the oscillatory insulin supply and the receptor dynamics can be of minute significance only. It is possible, however, to interpret seemingly conflicting results of clinical studies in terms of their different experimental conditions with respect to the hepatic glucose release. If this release is operating near an upper limit, an oscillatory insulin supply will be more efficient in lowering the blood glucose level than a constant supply. If the insulin level is high enough for the hepatic release of glucose to nearly vanish, the opposite effect is observed. For insulin concentrations close to the point of inflection of the insulin-glucose dose-response curve an oscillatory and a constant insulin infusion produce similar effects.

Intensive care of severely wounded military and civilian casualties in Zadar, Croatia.

From August 1, 1991, to May 30, 1992, 148 severely wounded military and civilian casualties with the injury severity score of 3 to 5 were treated in the intensive care unit of the Zadar General Hospital. There were 138 male and 10 female patients; their mean age was 32 years. There were 64 wounded civilians and 84 wounded soldiers. The average evacuation time was 3 hours. Twelve (8%) severely wounded persons died. The cause of death was craniocerebral injury in 7 patients (58%) and hemorrhage in 4 patients (33%). Complications following shock-like acute renal failure, gastrointestinal bleeding, coagulopathy, and hepathopathy developed in 18 wounded persons (12%).