Molecular cloning and characterization of Pals, proteins associated with mLin-7

In Caenorhabditis elegans, three PDZ domain proteins, Lin-2, Lin-7, and Lin-10, are necessary for the proper targeting of the Let-23 growth factor receptor to the basolateral surface of epithelial cells. It has been demonstrated that homologues of Lin-2, Lin-7, and Lin-10 form a heterotrimeric complex in mammalian brain. Using Far Western overlay assay, we have identified additional proteins that can bind to the amino terminus of mLin-7 and cloned the genes encoding these proteins using bacterial expression cloning. We call these proteins Pals, for proteins associated with Lin-7. These proteins, which include mammalian Lin-2, contain a conserved mLin-7 binding domain in addition to guanylate kinase, PDZ (postsynaptic density 95/discs large/zona occludens-1), and Src homology 3 domains. Using site-directed mutagenesis, we have identified the conserved residues among these proteins crucial for mLin-7 binding. Two of these proteins, Pals1 and Pals2, are newly described. Pals1 consists of 675 amino acids and maps to mouse chromosome 12. Pals2 was found to exist in two splice forms of 539 and 553 amino acids and maps to mouse chromosome 6. Like mLin-2, Pals1 and Pals2 localize to the lateral membrane in Madin-Darby canine kidney cells. Pals proteins represent a new subfamily of membrane-associated guanylate kinases that allow for multiple targeting complexes containing mLin-7.     

DWnt4 regulates cell movement and focal adhesion kinase during Drosophila ovarian morphogenesis

Cell motility is regulated by extracellular cues and by intracellular factors that accumulate at sites of contact between cells and the extracellular matrix. One of these factors, focal adhesion kinase (FAK), regulates the cycle of focal adhesion formation and disassembly that is required for cell movement to occur. Recently, Wnt signaling has also been implicated in the control of cell movement in vertebrates, but the mechanism through which Wnt proteins influence motility is unclear. We demonstrate that Drosphila Wnt4 is required for cell movement and FAK regulation during ovarian morphogenesis. Dfrizzled2, Disheveled, and protein kinase C are also required. The DWnt4 cell motility pathway is distinct from both the canonical Wnt pathway and the planar polarity pathway. Our data suggest that DWnt4 facilitates motility through regulation of focal adhesions.     

Structural comparison of fibroblast growth factor-specific heparan sulfates derived from a growing or differentiating neuroepithelial cell line

Heparan sulfate (HS) glycosaminoglycans are essential modulators of fibroblast growth factor (FGF) activity both in vivo and in vitro, and appear to act by cross-linking particular forms of FGF to appropriate FGF receptors. We have recently isolated and characterized two separate HS pools derived from immortalized embryonic day 10 mouse neuroepithelial 2.3D cells: one from cells in log growth phase, which greatly potentiates the activity of FGF-2, and the other from cells undergoing contact-inhibition and differentiation, which preferentially activates FGF-1. These two pools of HS have very similar functional activities to those species isolated from primary neuroepithelial cells at corresponding stages of active proliferation or differentiation. We present here a structural comparison between these cell line HS species to establish the nature of the changes that occur in the biosynthesis of HS. A combination of chemical and enzymatic cleavage, low pressure chromatography and strong anion-exchange HPLC were used to generate full chain models of each species. Overall, the HS pools synthesized in the dividing cell line pools possessed less complex sulfation than those derived from more differentiated, growth arrested cells.      

Structure/function analysis of the PII signal transduction protein of Escherichia coli: genetic separation of interactions with protein receptors.

The PII protein, encoded by glnB, is known to interact with three bifunctional signal transducing enzymes (uridylyltransferase/uridylyl-removing enzyme, adenylyltransferase, and the kinase/phosphatase nitrogen regulator II [NRII or NtrB]) and three small-molecule effectors, glutamate, 2-ketoglutarate, and ATP. We constructed 15 conservative alterations of PII by site-specific mutagenesis of glnB and also isolated three random glnB mutants affecting nitrogen regulation. The abilities of the 18 altered PII proteins to interact with the PII receptors and the small-molecule effectors 2-ketoglutarate and ATP were examined by using purified components. Results with certain mutants suggested that the specificity for the various protein receptors was altered; other mutations affected the interaction with all three receptors and the small-molecule effectors to various extents. The apex of the large solvent-exposed T loop of the PII protein (P. D. Carr, E. Cheah, P. M. Suffolk, S. G. Vasudevan, N. E. Dixon, and D. L. Ollis, Acta Crytallogr. Sect. D 52:93-104, 1996), which includes the site of PII modification, was not required for the binding of small-molecule effectors but was necessary for the interaction with all three receptors. Mutations altering residues of this loop or affecting the nearby B loop of PII, which line a cleft between monomers in the trimeric PII, affected the interactions with protein receptors and the binding of small-molecule ligands. Thus, our results support the predictions made from structural studies that the exposed loops of PII and cleft formed at their interface are the sites of regulatory interactions.     

Experience in the application of Java Technologies in telemedicine

Java language has been demonstrated to be an effective tool in supporting medical image viewing in Russia. This evaluation was completed by obtaining a maximum of 20 images, depending on the client's computer workstation from one patient using a commercially available computer tomography (CT) scanner. The images were compared against standard CT images that were viewed at the site of capture. There was no appreciable difference. The client side is a lightweight component that provides an intuitive interface for end users. Each image is loaded in its own thread and the user can begin work after the first image has been loaded. This feature is especially useful on slow connection speed, 9.6 Kbps for example. The server side, which is implemented by the Java Servlet Engine works more effective than common gateway interface (CGI) programs do. Advantages of the Java Technology place this program on the next level of application development. This paper presents a unique application of Java in telemedicine.     

Effects of age,replicative lifespan and growth rate of human nucleus pulposus cells on selecting age range for cell-based biological therapies for degenerative disc diseases

Autologous disc cell implantation, growth factors and gene therapy appear to be promising therapies for disc regeneration. Unfortunately, the replicative lifespan and growth kinetics of human nucleus pulposus (NP) cells related to host age are unclear. We investigated the potential relations among age, replicative lifespan and growth rate of NP cells, and determined the age range that is suitable for cell-based biological therapies for degenerative disc diseases. We used NP tissues classified by decade into five age groups: 30s, 40s, 50s, 60s and 70s. The mean cumulative population doubling level (PDL) and population doubling rate (PDR) of NP cells were assessed by decade. We also investigated correlations between cumulative PDL and age, and between PDR and age. The mean cumulative PDL and PDR decreased significantly in patients in their 60s. The mean cumulative PDL and PDR in the younger groups (30s, 40s and 50s) were significantly higher than those in the older groups (60s and 70s). There also were significant negative correlations between cumulative PDL and age, and between PDR and age. We found that the replicative lifespan and growth rate of human NP cells decreased with age. The replicative potential of NP cells decreased significantly in patients 60 years old and older. Young individuals less than 60 years old may be suitable candidates for NP cell-based biological therapies for treating degenerative disc diseases.