Single molecule binding dynamics measured with atomic force microscopy.

We present a new method to analyse simultaneous Topography and RECognition Atomic Force Microscopy data such that it becomes possible to measure single molecule binding rates of surface bound proteins. We have validated this method on a model system comprising a S-layer surface modified with Strep-tagII for binding sites and strep-tactin bound to an Atomic Force Microscope tip through a flexible Poly-Ethylene-Glycol linker. At larger distances, the binding rate is limited by the linker, which limits the diffusion of the strep-tactin molecule, but at lateral distances below 3 nm, the binding rate is solely determined by the intrinsic molecular characteristics and the surface geometry and chemistry of the system. In this regime, Kon as determined from single molecule TREC data is in agreement with Kon determined using traditional biochemical methods.

Imaging and detection of single molecule recognition events on organic semiconductor surfaces.

The combination of organic thin film transistors and biological molecules could open new approaches for the detection and measurement of properties of biological entities. To generate specific addressable binding sites on such substrates, it is necessary to determine how single biological molecules, capable of serving as such binding sites behave upon attachment to semiconductor surfaces. Here, we use a combination of high-resolution atomic force microscopy topographical imaging and single molecule force spectroscopy (TREC), to study the functionality of antibiotin antibodies upon adsorption on pentacene islands, using biotin-functionalized, magnetically coated AFM tips. The antibodies could be stably adsorbed on the pentacene, preserving their functionality of recognizing biotin over the whole observation time of more than one hour. We have resolved individual antigen binding sites on single antibodies for the first time. This highlights the resolution capacity of the technique.

Role for retroperitoneal lymphadenectomy for testis cancer.

There continue to be several controversies surrounding the role for retroperitoneal lymphadenectomy (RPL) in the management of patients with germ cell cancer of the testis. The initial treatment options for those with clinical stage I disease are surveillance (orchiectomy only), RPL or chemotherapy. Survival rates are similar with RPL and surveillance. Surgical morbidity has been reduced as techniques for RPL continue to improve. The likelihood of early or late (> 2 years) recurrence in the retroperitoneum is almost eliminated by RPL. Fewer follow-up computerized tomography scans of the abdomen are required and there are opportunities to reduce the duration and methods of follow-up, compared with surveillance. For patients with stage II disease, chemotherapy and RPL are equally effective initial treatment options but many patients require a combined approach. Initial RPL should be reserved for patients with smaller volume disease and possibly with lower preoperative marker levels. With RPL, patients are accurately staged and cured most of the time without double treatment. Approximately 30% of those with larger masses will have residual disease after initial chemotherapy and will require RPL as a second treatment. The third indication for RPL is to excise residual retroperitoneal masses following primary chemotherapy. Models to predict the presence of residual viable tumor, rather than necrosis only, at the time of surgery have been developed. If the orchiectomy specimen contained no teratoma, the tumor markers normalize after three or four courses of chemotherapy, and if the residual mass on computerized tomography scan is less than 2 cm in diameter, the rate of viable tumor may be low enough to omit RPL. In this way, the greater morbidity often associated with post-chemotherapy RPL may be avoided.

Tissue distribution of ryanodine receptor isoforms and alleles determined by reverse transcription polymerase chain reaction.

The tissue distribution of mRNA for ryanodine receptor (ryr) isoforms in various porcine tissues has been determined using the reverse transcription-polymerase chain reaction (RT-PCR). First strand cDNA was synthesized from total tissue RNA with reverse transcriptase and random hexamer primers. PCR primers were selected to amplify an approximately 500-base pair segment from homologous regions near the 5' end of the skeletal (ryr1), cardiac (ryr2), or brain (ryr3) ryr cDNA sequences. The specific amplification of each of the ryr isoforms was confirmed by restriction enzyme mapping and DNA sequencing. A ryr1 RT-PCR product was identified in skeletal muscle and esophagus, a ryr2 RT-PCR product was identified in cardiac muscle, aorta and esophagus, and a ryr3 RT-PCR product was identified in skeletal and cardiac muscle, aorta, esophagus, adrenal gland, small intestine, and lung. All three ryr isoforms were identified throughout the brain, including the parietal, frontal, and temporal lobes of the cerebrum, thalamus/hypothalamus, cerebellum, and brain stem. The normal (Arg615) and mutant (Cys615) ryr1 alleles were expressed in the brains of normal and malignant hyperthermia susceptible pigs, respectively. These results thus demonstrate expression of two ryr isoforms in each type of striated muscle, and all ryr isoforms in a number of regions of the nervous system. The wide distribution of ryr1 in the brain provides a possible neurogenic etiology of malignant hyperthermia.