Kinetic Process of β-Amyloid Formation via Membrane Binding

Recently we have studied thermodynamics of membrane-mediated β-amyloid formation in equilibrium experiments using penetratin-lipid mixtures. The results showed that penetratin bound to the membrane interface in the α-helical conformation when the peptide/lipid (P/L) ratios were below a lipid-dependent critical value P/L^∗. When P/L reached P/L^∗, small β-aggregates emerged, which served as the nuclei for large β-aggregates. Here we studied the corresponding kinetic process to understand the potential barriers for the membrane-mediated β-amyloid formation. We performed kinetic experiments using giant unilamellar vesicles made of 7:3 DOPC/DOPG. The observed time behavior of individual giant unilamellar vesicles, although complex, exhibited the physical effects seen in equilibrium experiments. Most interestingly, a potential barrier appeared to block penetratin from translocating across the bilayer. As a result, the kinetic value for the critical threshold P/L^∗ is roughly one-half of the value measured in equilibrium where peptides bind symmetrically on both sides of lipid bilayers. We also investigated the similarity and differences between the charged and neutral lipids in their interactions with penetratin. We reached an important conclusion that the bound states of peptides in lipid bilayers are largely independent of the charge on the lipid headgroups.     

Enhancement of cell growth by intracellularly expressed herpes simplex virus thymidine kinase

A recombinant cell line (NIH3T3:pLtkSN) was made by infecting parental cells (NIH3T3) with a recombinant retrovirus (pLtkSN) encoding herpes simplex virus thymidine kinase (HSVtk) gene. The cells expressing HSVtk (NIH3T3:pLtkSN) grew 2.3 times more than the parental cells (NIH3T3) in Dulbecco's Modified Eagles Media containing 10% (v/v) horse serum. The NIH3T3:pLtkSN cells also showed a significant enhancement in the maximal cell concentration and the specific growth rate even at 2.5% serum concentration. The specific O2 uptake rate of NIH3T3 was 2.1 times greater than that of NIH3T3:pLtkSN. Under both O2-limited and O2-unlimited conditions, it appears that HSVtk plays an important role in enhancing the growth characteristics of animal cells.     

A simple method using PCR for direct sequencing of genomic DNA from frozen tumor tissue embedded in optimal cutting temperature compound.

Described here is a three-day protocol that directly yields DNA sequence after isolating and PCR amplifying genomic DNA from a small sample of frozen nasopharyngeal carcinoma tissue embedded in optimal cutting temperature (OCT) compound. The method is consistently successful, reproducible and will facilitate the rapid analysis of DNA sequence from very small samples.     

Corolla wilting facilitates delayed autonomous self-pollination in Pedicularis dunniana (Orobanchaceae)

Structural changes associated with corolla wilting may serve as a mechanism for effecting self-pollination. Low pollinator visitation, high seed production and a corolla that persists after anthesis indicates that Pedicularis dunniana is autogamous. Delayed autonomous self-pollination is facilitated by corolla wilting. Wilting of the upper lip (galea) brought the pollen laden anthers into contact with the stigma resulting in the deposition of self pollen on the stigma. The seed set of flowers either emasculated, or with restrained galeae thus preventing anthers brushing against the stigma, was significantly lower than that of open-pollinated flowers. This demonstrates that autogamy occurs in this species through corolla wilting. Germination experiments indicated that outcross seedlings were more vigorous than selfed seedlings as a result of inbreeding depression. It is likely that autogamy provides reproductive assurance for P. dunniana under conditions of pollinator scarcity.     

Observing the Temperature Dependent Transition of the GP2 Peptide Using Terahertz Spectroscopy

The GP2 peptide is derived from the Human Epidermal growth factor Receptor 2 (HER2/nue), a marker protein for breast cancer present in saliva. In this paper we study the temperature dependent behavior of hydrated GP2 at terahertz frequencies and find that the peptide undergoes a dynamic transition between 200 and 220 K. By fitting suitable molecular models to the frequency response we determine the molecular processes involved above and below the transition temperature (T _D). In particular, we show that below T _D the dynamic transition is dominated by a simple harmonic vibration with a slow and temperature dependent relaxation time constant and that above T _D, the dynamic behavior is governed by two oscillators, one of which has a fast and temperature independent relaxation time constant and the other of which is a heavily damped oscillator with a slow and temperature dependent time constant. Furthermore a red shifting of the characteristic frequency of the damped oscillator was observed, confirming the presence of a non-harmonic vibration potential. Our measurements and modeling of GP2 highlight the unique capabilities of THz spectroscopy for protein characterization.