A novel preclinical model of human malignant melanoma utilizing bioreactor rotating-wall vessels

Malignant melanoma poses a serious health risk which is becoming more crucial as the incidence of this disease steadily increases. The development of appropriate in vitro models that reflect the in vivo tumor environment is a key factor for the study of this malignancy. The local tumor microenvironment plays a critical role in the ability of tumor cells to proliferate and metastasize. While interactions among various cell types are known to be important for tumor growth, most in vitro models utilize only tumor cells, ignoring the importance of tumor-stroma interactions, as well as the contribution of immune cells, which may be important for potential therapies. In addition, the cellular architecture found in vivo, known to be involved in changes in gene expression, is not reflected in standard two-dimensional culture systems. In this study, we have utilized rotating-vessel bioreactors to culture minced human melanoma specimens, allowing the culture of three-dimensional structures which reflect the cellular architecture and heterogeneous composition of the tumor site in vivo. The viability of the pieces in culture can be maintained for 1-2 wk. Immunohistochemical analysis shows multiple cellular types similar to the in vivo situation. Therefore, this system provides a unique model of human melanoma that mimics the in vivo tumor environment much more closely than current culture methods. This novel system may be utilized to determine the mechanism of action of current therapy protocols, as well as to develop new treatment regimens.     

Slow Relaxation Process in DNA

A dynamic transition at temperatures 200–230K is observed in manyhydrated bio-polymers. It shows up as a sharp increase of the mean-squaredatomic displacements above this temperature range. We present neutronscattering data of DNA at different levels of hydration. The analysis showsthat the dynamic transition in DNA is related to a slow relaxation processin the MHz-GHz frequency range. This slow relaxation process iscompletely suppressed in the dry DNA sample where no dynamic transitionwas observed. The nature of the slow process is discussed. We ascribe it toa global relaxation of DNA molecule that involves cooperative motion ofmany base-pairs and backbone.     

Design and simple routes of synthesis of oligonucleotide conjugates for studies of DNA triple helix formation

A series of oligonucleotides conjugated to intercalators, as well as fluorescent and lipophilic substances, minor groove binders and photoactive molecules were synthesized for studies of their ability to form a stable triple helix. Purine-rich short double stranded DNA fragments from HIV-1 genome and pyrimidine 16-mer oligodeoxyribonucleotide were used as models. A conjugate of a dipyrido[3,2-a:2',3'-c]phenazine-ruthenium (II) complex and a triple helix-forming oligonucleotide was constructed. Upon sequence-specific duplex and triplex formation of the conjugate, the ruthenium complex becomes highly fluorescent. The attached ruthenium complex induces a stabilization of the DNA triple helix and a significant increase of the time of residence of the third strand on the duplex.     

Slow rate during AF improves ventricular performance by reducing sensitivity to cycle length irregularity

Atrial fibrillation (AF) is characterized by short and irregular ventricular cycle lengths (VCL). While the beneficial effects of heart rate slowing (i.e., the prolongation of VCL) in AF are well recognized, little is known about the impact of irregularity. In 10 anesthetized dogs, R-R intervals, left ventricular (LV) pressure, and aortic flow were collected for >500 beats during fast AF and when the average VCL was prolonged to 75%, 100%, and 125% of the intrinsic sinus cycle length by selective atrioventricular (AV) nodal vagal stimulation. We used the ratio of the preceding and prepreceding R-R intervals (RR(p)/RR(pp)) as an index of cycle length irregularity and assessed its effects on the maximum LV power, the minimum of the first derivative of LV pressure, and the time constant of relaxation by using nonlinear fitting with monoexponential functions. During prolongation of VCL, there was a pronounced decrease in curvature with the formation of a plateau, indicating a lesser dependence on RR(p)/RR(pp). We conclude that prolongation of the VCL during AF reduces the sensitivity of the LV performance parameters to irregularity.     

Crystal structures and enzymatic properties of three formyltransferases from archaea: environmental adaptation and evolutionary relationship

Formyltransferase catalyzes the reversible formation of formylmethanofuran from N(5)-formyltetrahydromethanopterin and methanofuran, a reaction involved in the C1 metabolism of methanogenic and sulfate-reducing archaea. The crystal structure of the homotetrameric enzyme from Methanopyrus kandleri (growth temperature optimum 98 degrees C) has recently been solved at 1.65 A resolution. We report here the crystal structures of the formyltransferase from Methanosarcina barkeri (growth temperature optimum 37 degrees C) and from Archaeoglobus fulgidus (growth temperature optimum 83 degrees C) at 1.9 A and 2.0 A resolution, respectively. Comparison of the structures of the three enzymes revealed very similar folds. The most striking difference found was the negative surface charge, which was -32 for the M. kandleri enzyme, only -8 for the M. barkeri enzyme, and -11 for the A. fulgidus enzyme. The hydrophobic surface fraction was 50% for the M. kandleri enzyme, 56% for the M. barkeri enzyme, and 57% for the A. fulgidus enzyme. These differences most likely reflect the adaptation of the enzyme to different cytoplasmic concentrations of potassium cyclic 2,3-diphosphoglycerate, which are very high in M. kandleri (>1 M) and relatively low in M. barkeri and A. fulgidus. Formyltransferase is in a monomer/dimer/tetramer equilibrium that is dependent on the salt concentration. Only the dimers and tetramers are active, and only the tetramers are thermostable. The enzyme from M. kandleri is a tetramer, which is active and thermostable only at high concentrations of potassium phosphate (>1 M) or potassium cyclic 2,3-diphosphoglycerate. Conversely, the enzyme from M. barkeri and A. fulgidus already showed these properties, activity and stability, at much lower concentrations of these strong salting-out salts.     

Effects of simulated microgravity on thyroid carcinoma cells

We aimed to investigate whether simulated microgravity on thyroid carcinoma cells could help to perform in vitro cancer studies such as antitumor drug tests more reliable and to spare animal experiments. We cultured cancer cells at 0 g to enable formation of three-dimensional multicellular tumor spheroids (MCTS), which will resemble the originating tumors. Under microgravity human follicular cells (ML-1 cell line) keep floating with-out stirring so that initial cell-cell interactions required for spheroid formation will be induced by forces due to biochemical components actually expressed on surfaces of cells, whereas gravity related push- or shear events will not influence MCTS formation. Within 12 hours of clinorotation the monolayer turned spontaneously into MCTS with remarkable features: An increase of extracellular matrix proteins and TGF-beta 1. Thyroglobulin, ft3 and ft4 secretion were markedly reduced. These data are in agreement with the observation that astronauts show low thyroid hormone levels after spaceflight.     

Fitting manifold surfaces to three-dimensional point clouds

We present a technique for fitting a smooth, locally parameterized surface model (called the manifold surface model) to unevenly scattered data describing an anatomical structure. These data are acquired from medical imaging modalities such as CT scans or MRI. The manifold surface is useful for problems which require analyzable or parametric surfaces fitted to data acquired from surfaces of arbitrary topology (e.g., entire bones). This surface modeling work is part of a larger project to model and analyze skeletal joints, in particular the complex of small bones within the wrist and hand. To demonstrate the suitability of this model we fit to several different bones in the hand, and to the same bone from multiple people.     

A novel GlcNAcalpha1-HPO3-6Gal(1-1)ceramide antigen and alkylated inositol-phosphoglycerolipids expressed by the liver fluke Fasciola hepatica

The acidic (glyco)lipids of the parasitic liver fluke Fasciola hepatica exhibited two different phosphate-containing species, designated AL-I and AL-II, which were analyzed by MALDI-TOF MS, ESI MS, NMR, methylation analysis, and combined GC-MS in conjunction with HF treatment. AL-I was structurally determined as 1-O-hexadecyl-sn-glycerol-3-phosphoinositol, an ether bond variant of lysophosphatidylinositol. The structure of AL-II was shown to be GlcNAcalpha1-HPO3-6Gal(1-1)ceramide. Ceramide analysis revealed as major components 2-hydroxyoctadecanoic acid [18:0(2-OH)] together with C18- and C20-phytosphingosines. AL-II was apparently highly antigenic and strongly recognized by both animal- and human-F. hepatica infection sera. Furthermore, inhibition ELISAs revealed that the unusual antigenic determinant GlcNAcalpha1-HPO3- phosphate might have a potential in the serodiagnosis of F. hepatica infections.