Transients in acetylcholine receptor site density and degradation during reinnervation of mouse sternomastoid muscle

The degradation rates of acetylcholine receptors (AchRs) were evaluated at the neuromuscular junction during and just after reinnervation of denervated muscles. When mouse sternomastoid muscles are denervated by multiple nerve crush, reinnervation begins 2-4 days later and is complete by day 7-9 after the last crush. In fully innervated muscles, the AChR degradation rate is stable and slow (t1/2 approximately 10 days), whereas after denervation the newly inserted receptors degrade rapidly (t1/2 approximately 1.2 days). The composite profile of degradation, which a mixture of the stable and the rapid receptors would give, is not observed during reinnervation. Instead, the receptors inserted between 2.5 and 7.5 days after the last crush all have an intermediate degradation rate of t1/2 approximately 3.7 days with standard error +/- 0.3 days. The total receptor site density at the endplate was evaluated during denervation and during reinnervation. As predicted theoretically, the site density increased substantially, but temporarily, after denervation. An analogous deleterious substantial decrease in density would be expected during reinnervation, without the intermediate receptor. This decrease is not observed, however, because of a large insertion rate at intermediate times (3000 +/- 700 receptor complexes per micro m2 per day). The endplate density of receptors thus remains relatively constant.     

Cla4 protein kinase is essential for filament formation and invasive growth of Yarrowia lipolytica

The non-conventional yeast Yarrowia lipolytica is a suitable model for the study of yeast dimorphism. In order to identify genes that may be involved in the regulation of this process, random mutagenesis was performed. This led to the isolation of monomorphic mutants that had lost the ability to grow in a hyphal form both in liquid and on solid medium. Filamentation was restored to one of the mutants by transformation with a fragment of Y. lipolytica genomic DNA containing a single 2766-bp ORF. The predicted protein has a molecular weight of 99.6 kDa and is highly homologous to the protein kinases Cla4 of Candida albicans and Saccharomyces cerevisiae, which are members of the p21-activated kinase (PAK) family. Analysis of the putative protein sequence identified conserved C-terminal catalytic, and internal Cdc42p-binding regions, as well as a pleckstrin homology domain typical of PAK kinases. The results indicate that CLA4 is a single-copy gene located on the chromosome V of Y. lipolytica. Deletion of CLA4 is not lethal, but completely eliminates the ability to form filaments and to invade agar. A strain lacking a functional CLA4 gene exhibits an aberrant distribution of chitin in the cell wall, indicating a possible role for the Cla4 protein kinase in the maintenance of cell polarity in Y. lipolytica.     

Mechanotransduction through the cytoskeleton

We constructed a model cytoskeleton to investigate the proposal that this interconnected filamentous structure can act as a mechano- and signal transducer. The model cytoskeleton is composed of rigid rods representing actin filaments, which are connected with springs representing cross-linker molecules. The entire mesh is placed in viscous cytoplasm. The model eukaryotic cell is submitted to either shock wave-like or periodic mechanical perturbations at its membrane. We calculated the efficiency of this network to transmit energy to the nuclear wall as a function of cross-linker stiffness, cytoplasmic viscosity, and external stimulation frequency. We found that the cytoskeleton behaves as a tunable band filter: for given linker molecules, energy transmission peaks in a narrow range of stimulation frequencies. Most of the normal modes of the network are spread over the same frequency range. Outside this range, signals are practically unable to reach their destination. Changing the cellular ratios of linker molecules with different elastic characteristics can control the allowable frequency range and, with it, the efficiency of mechanotransduction.     

Multivariate approach for selecting sets of differentially expressed genes

An important problem addressed using cDNA microarray data is the detection of genes differentially expressed in two tissues of interest. Currently used approaches ignore the multidimensional structure of the data. However it is well known that correlation among covariates can enhance the ability to detect less pronounced differences. We use the Mahalanobis distance between vectors of gene expressions as a criterion for simultaneously comparing a set of genes and develop an algorithm for maximizing it. To overcome the problem of instability of covariance matrices we propose a new method of combining data from small-scale random search experiments. We show that by utilizing the correlation structure the multivariate method, in addition to the genes found by the one-dimensional criteria, finds genes whose differential expression is not detectable marginally.     

Functional characterization of two novel mammalian electrogenic proton-dependent amino acid cotransporters

We cloned two cDNAs encoding proton/amino acid cotransporters, designated as mPAT1 and mPAT2, from murine tissues. They were identified by sequence similarity to the amino acid/auxin permease family member of lower eukaryotes. We functionally characterized both transporters by flux studies and electrophysiology after expression in Xenopus laevis oocytes. Both mPAT1 and mPAT2 induced a pH-dependent electrogenic transport activity for small amino acids (glycine, alanine, and proline) that is altered by membrane potential. Direct evidence for amino acid/H(+)-symport was shown by intracellular acidification, and a flux coupling stoichiometry for proline/H(+)-symport of 1:1 was determined for both transporters. Besides small apolar L-amino acids, the transporters also recognize their D-enantiomers and selected amino acid derivatives such as gamma-aminobutyric acid. The mPAT1 transporter, the murine orthologue of the recently cloned rat LYAAT-1 transporter, can be considered as a low affinity system when compared with mPAT2. The mRNA of mPAT1 is highly expressed in small intestine, colon, kidney, and brain; the mPAT2-mRNA is mainly found in heart and lung. Phenotypically, the PAT1 transporter possesses the same functional characteristics as the previously described proton-dependent amino acid transport process in apical membranes of intestinal and renal epithelial cells.     

Shedding of the interleukin-6 (IL-6) receptor (gp80) determines the ability of IL-6 to induce gp130 phosphorylation in human osteoblasts

Human osteoblasts produce interleukin-6 (IL-6) and respond to IL-6 in the presence of soluble IL-6 receptor (sIL-6R), but the cell surface expression of IL-6R and the mechanism of sIL-6R production are largely unknown. Three different human osteoblast-like cell lines (MG-63, HOS, and SaOS-2) and bone marrow-derived primary human osteoblasts expressed both IL-6R and gp130 as determined by flow cytometry and immunoprecipitation. However, the membrane-bound IL-6R was nonfunctional, as significant tyrosine phosphorylation of gp130 did not occur in the presence of IL-6. Phorbol myristate acetate induced a dramatic increase of both IL-6R shedding (i.e. the production of sIL-6R) and IL-6 release in osteoblast cultures, but the cell surface expression of gp130 remained unchanged. IL-6 complexed with sIL-6R, either exogenously introduced or derived from the nonfunctional cell surface form by shedding, induced rapid tyrosine phosphorylation of gp130. This effect was inhibited by neutralizing antibodies to either sIL-6R or gp130, indicating that the gp130 activation was induced by IL-6/sIL-6R/gp130 interaction. Protein kinase C inhibitors blocked phorbol myristate acetate-induced and spontaneous shedding of IL-6R resulting in the absence of sIL-6R in the culture medium, which in turn also prevented the activation of gp130. In conclusion, human osteoblasts express cell surface IL-6R, which is unable to transmit IL-6-induced signals until it is shed into its soluble form. This unique mechanism provides the flexibility for osteoblasts to control their own responsiveness to IL-6 via the activation of an IL-6R sheddase, resulting in an immediate production of functionally active osteoblast-derived sIL-6R.     

STRP Screening Sets for the human genome at 5 cM density

Background  

Short tandem repeat polymorphisms (STRPs) are powerful tools for gene mapping and other applications. A STRP genome scan of 10 cM is usually adequate for mapping single gene disorders. However mapping studies involving genetically complex disorders and especially association (linkage disequilibrium) often require higher STRP density.     

Low power millimeter wave irradiation exerts no harmful effect on human keratinocytes in vitro

Low power millimeter wave (LP-MW) irradiation has been successfully used in clinical practice as an independent and/or supplemental therapy in patients with various diseases. It is still not clear, however, whether exposed skin is directly affected by repeated LP-MW irradiation and whether cells of the epidermis can be activated by the absorbed energy. Keratinocytes, the most numerous component of the epidermis are believed to manifest functional responses to physical stimuli. In this study we analyzed whether LP-MW irradiation modulated the production of chemokines, including RANTES and IP-10 of keratinocytes in vitro. We also investigated whether LP-MW irradiation induces a heat stress reaction in keratinocytes, and stimulates heat shock protein 70 (Hsp70) production. Vital staining of keratinocytes with carboxyfluorescein succinimidyl ester and ethidium bromide was used to analyze the MW effect on the viability of adherent cells. In addition, we studied the effect of LP-MW irradiation on intercellular gap junctional communication in keratinocyte monolayers by Lucifer yellow dye transfer. We found no significant changes in constitutive RANTES and inducible IP-10 production following LP-MW irradiation. LP-MW exposure of keratinocyte monolayers did not alter Hsp70 production, unlike exposure to higher power MWs (HP-MW) or hyperthermia (43 degrees C; 1 h). LP-MW irradiation and hyperthermia did not alter the viability of adherent keratinocytes, while HP-MW irradiation induced cellular damage within the beam area. Finally, we found no alteration in the gap junctional intercellular communication of keratinocytes following LP-MW irradiation, which on the other hand, was significantly increased by hyperthermia. In summary, we detected no harmful effect of LP-MW irradiation on both keratinocyte function and structure in vitro, although these cells were sensitive to higher MW power that developed heat stress reaction and cellular damage. Our results provide further evidence that LP-MW irradiation does not induce evidence of skin inflammation or keratinocyte damage and that its clinical application appears to be safe.